Constructs for expressing transgenes using regulatory elements from brachypodium ubiquitin 1C genes

ABSTRACT

Provided are constructs and methods for expressing a transgene in plant cells and/or plant tissues using the regulatory elements, including the promoters and/or 3′-UTRs, isolated from  Brachypodium distachyon  ubiquitin genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/872,134, filed Aug. 30, 2013, which is hereby incorporated byreference in its entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 64 KB ASCII (Text) file named231412SubSeqListing.txt, created on Sep. 7, 2016.

BACKGROUND

Plant transformation is an attractive technology for use in introducingagronomically desirable traits or characteristics into different cropplant species. Plant species are developed and/or modified to haveparticular desirable traits. Generally, desirable traits include, forexample, improving nutritional value quality, increasing yield,conferring pest or disease resistance, increasing drought and stresstolerance, improving horticultural qualities (e.g., pigmentation andgrowth), imparting herbicide resistance, enabling the production ofindustrially useful compounds and/or materials from the plant, and/orenabling the production of pharmaceuticals.

Transgenic plants comprising multiple transgenes stacked at a singlegenomic locus are produced via plant transformation technologies. Planttransformation technologies result in the introduction of a transgeneinto a plant cell, recovery of a fertile transgenic plant that containsthe stably integrated copy of the transgene in the plant genome, andsubsequent transgene expression via transcription and translation of thetransgene(s) results in transgenic plants that possess desirable traitsand phenotypes. Each transgene in a stack typically requires anindependent promoter for gene expression, and thus multiple promotersare used in a transgene stack.

The need for co-expression of multiple transgenes for regulating thesame trait frequently results in the repeated use of the same promoterto drive expression of the multiple transgenes. However, the repeateduse of promoters comprising sequences that share a high level ofsequence identity may lead to homology-based gene silencing (HBGS). HBGShas been observed to occur frequently in transgenic plants (Peremarti etal., 2010) when repetitive DNA sequences are used within a transgene. Inaddition, repeated use of similar DNA sequences in transgene constructshas proven to be challenging in Agrobacterium due to recombination andinstability of the plasmid.

Described herein are ubiquitin regulatory elements (e.g., promoters and3′-UTR) that share low levels of sequence identity or homology with theMaize ubiquitin) promoter. Further described are constructs and methodsutilizing ubiquitin regulatory elements.

SUMMARY

Disclosed herein are constructs and methods for expressing a transgenein plant cells and/or plant tissues. In one embodiment regulatoryelements of a ubiquitin gene are purified from Panicum virgatum,Brachypodium distachyon, or Setaria italica genomes and recombined withsequences not natively linked to the regulatory elements to create anexpression vector for expressing transgenes in plant cells not native tothe ubiquitin regulatory sequences. In one embodiment an expressionvector is provided wherein the regulatory elements of a ubiquitin geneare operably linked to a polylinker sequence. Such an expression vectoreases the insertion of a gene or gene cassette into the vector in anoperably linked state with the ubiquitin gene regulatory sequences.

In an embodiment, a construct is provided comprising a Panicum virgatum,Brachypodium distachyon, or Setaria italica ubiquitin promoter. In anembodiment, a gene expression cassette is provided comprising a Panicumvirgatum, Brachypodium distachyon or Setaria italica ubiquitin promoteroperably linked to a transgene. In an embodiment, a gene expressioncassette includes a Panicum virgatum, Brachypodium distachyon or Setariaitalica ubiquitin 5′-UTR operably linked to a transgene. In anembodiment, a gene expression cassette includes a Panicum virgatum,Brachypodium distachyon or Setaria italica ubiquitin 5′-UTR operablylinked to a promoter. In an embodiment, a gene expression cassetteincludes a Panicum virgatum, Brachypodium distachyon or Setaria italicaubiquitin intron operably linked to a transgene. In an embodiment, agene expression cassette includes a Panicum virgatum, Brachypodiumdistachyon or Setaria italica ubiquitin intron operably linked to apromoter. In an embodiment, a construct includes a gene expressioncassette comprising Panicum virgatum, Brachypodium distachyon or Setariaitalica ubiquitin 3′-UTR. In an embodiment, a gene expression cassetteincludes Panicum virgatum, Brachypodium distachyon or Setaria italicaubiquitin 3′-UTR operably linked to a transgene. In an embodiment, agene expression cassette includes at least one, two, three, five, six,seven, eight, nine, ten, or more transgenes.

In an embodiment, a gene expression cassette includes independently a) aPanicum virgatum, Brachypodium distachyon, or Setaria italica ubiquitinpromoter, b) a Panicum virgatum, Brachypodium distachyon or Setariaitalica ubiquitin intron, c) a Panicum virgatum, Brachypodium distachyonor Setaria italica ubiquitin 5′-UTR, and d) a Panicum virgatum,Brachypodium distachyon or Setaria italica ubiquitin 3′-UTR.

In accordance with one embodiment a nucleic acid vector is providedcomprising a promoter operably linked to a non-ubiquitin transgene,wherein the promoter consists of SEQ ID NO: 15 or a sequence having 90%sequence identity with SEQ ID NO: 15. In a further embodiment thenucleic acid vector comprises a gene cassette, wherein the gene cassettecomprises a promoter, a non-ubiquitin transgene and a 3′ untranslatedregion, wherein the promoter consists of SEQ ID NO: 15 operably linkedto a first end of a transgene, wherein the second end of the transgeneis operably linked to a 3′ untranslated sequence consisting of SEQ IDNO: 4.

Methods of growing plants expressing a transgene using the Panicumvirgatum, Brachypodium distachyon, or Setaria italica promoters,5′-UTRs, introns, and 3′-UTRs are disclosed herein. Methods of culturingplant tissues and cells expressing a transgene using the Panicumvirgatum, Brachypodium distachyon or Setaria italica promoters, 5′-UTRs,introns, and 3′-UTRs are also disclosed herein.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided comprising a promoter operably linked to a non-ubiquitintransgene, wherein the promoter comprises SEQ ID NO: 1. In accordancewith one embodiment a non-Brachypodium plant or plant cell is providedcomprising SEQ ID NO: 1, or a sequence that has 90% sequence identitywith SEQ ID NO: 1 operably linked to a transgene. In one embodiment theplant is a corn variety. In one embodiment a plant, plant tissue, orplant cell is provided comprising a promoter operably linked to anon-ubiquitin transgene, wherein the promoter consists of SEQ ID NO: 15or 40. In one embodiment a non-Brachypodium plant or plant cell isprovided comprising a gene cassette, wherein the gene cassette comprisesa promoter operably linked to a transgene, further wherein the promoterconsists SEQ ID NO: 15. In a further embodiment the promoter is operablylinked to a first end of a transgene, wherein the second end of thetransgene is operably linked to a 3′ untranslated sequence consisting ofSEQ ID NO: 4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the protein alignment of Zea mays ubiquitin (ZM Ubi1)protein sequence to Brachypodium distachyon and Setaria italic ubiquitinsequences used for promoter identification. The Zm Ubi1 protein sequenceis disclosed herein as SEQ ID NO:22. The S. italica Ubi2 proteinsequence is disclosed herein as SEQ ID NO:23. The B. distachyon Ubi1promoter sequence is disclosed herein as SEQ ID NO:24. The B. distachyonUbi1C protein sequence is disclosed herein as SEQ ID NO:25. Theconsensus sequence is disclosed herein as SEQ ID NO:26.

FIG. 2 shows the alignment of Zea mays ubiquitin (ZM Ubi1) promoterpolynucleotide sequence to Brachypodium distachyon and Setaria italicaubiquitin promoter polynucleotides identified herein. The Zea mayUbiquitin 1 (Zm-Ubi) promoter sequence is disclosed herein as SEQ IDNO:27. The B. distachyon Ubi1 promoter sequence is disclosed herein asSEQ ID NO:16. The B. distachyon Ubi1-C promoter sequence is disclosedherein as SEQ ID NO:15. The S. italica Ubi2 promoter sequence isdisclosed herein as SEQ ID NO:17.

FIG. 3 is a plasmid map showing the synthesized Setaria italicaUbiquitin2 promoter genetic element.

FIG. 4 is a plasmid map showing the synthesized Brachypodium distachyonUbiquitin1 C promoter genetic element and flanking seamless cloningoverhang location.

FIG. 5 is a plasmid map showing the synthesized Brachypodium distachyonUbiquitin1 promoter genetic element and flanking seamless cloningoverhang location.

FIG. 6 is a plasmid map showing the expression vector containing Setariaitalica ubiquitin2 (SI-Ubi2) promoter fused to PhiYFP reporter gene.

FIG. 7 is a plasmid map showing the expression vector containingBrachypodium distachyon Ubiquitin) C promoter fused to PhiYFP reportergene.

FIG. 8 is a plasmid map showing the expression vector containingBrachypodium distachyon Ubiquitin1 promoter fused to PhiYFP reportergene.

FIG. 9 is a plasmid map showing the expression vector containing OS Act1(Rice Actin1) promoter fused to PhiYFP reporter gene.

FIG. 10 is a plasmid map showing the expression vector containing ZMUbi1 promoter fused to PhiYFP reporter gene.

FIG. 11 is a plasmid map showing the binary destination vector used tobuild binary expression vectors using Gateway technology.

FIG. 12 is a plasmid map showing the binary expression vector containingSetaria italica Ubiquitin2 (SI-Ubi2) promoter fused to yellowfluorescent protein (Phi YFP) marker gene coding region containingST-LS1 intron followed by fragment comprising a StPinII 3′UTR frompotato.

FIG. 13 is a plasmid map showing the binary expression vector containingBrachypodium distachyon Ubiquitin1 C promoter fused to yellowfluorescent protein (Phi YFP) marker gene coding region containingST-LS1 intron followed by fragment comprising a StPinII 3′UTR frompotato.

FIG. 14 is a plasmid map showing the binary expression vector containingBrachypodium distachyon Ubiquitin1 promoter fused to yellow fluorescentprotein (Phi YFP) marker gene coding region containing ST-LS1 intronfollowed by fragment comprising a StPinII 3′UTR from potato.

FIG. 15 is a plasmid map showing the binary expression vector containingOS Act1 promoter fused to yellow fluorescent protein (Phi YFP) markergene coding region containing ST-LS1 intron followed by fragmentcomprising a StPinII 3′UTR from potato.

FIG. 16 is a plasmid map showing the binary expression vector containingZM Ubi1 promoter fused to yellow fluorescent protein (Phi YFP) markergene coding region containing ST-LS 1 intron followed by fragmentcomprising a StPinII 3′UTR from potato.

FIG. 17 shows YFP expression in a T₀ leaf where YFP is driven by thecross species ubiquitin and Os Act 1 promoters as depicted in FIGS. 12,13, 14, 15, and 16.

FIG. 18 shows AAD1 expression in a T₀ leaf where AAD1 is driven by theZm Ubi 1 promoter as depicted in FIGS. 12, 13, 14, 15, and 16.

FIG. 19 shows transient YFP expression driven by the Brachypodiumdistachyon and Setaria italica novel promoters as compared to YFPexpression driven by the ZM Ubi1 and OS Act1 promoters.

FIG. 20 shows YFP expression in calli tissues driven by the novelBrachypodium distachyon and Setaria italica promoters as compared to YFPexpression driven by the ZM Ubi1 and OS Act1 promoters.

FIG. 21 shows YFP expression in root tissue driven by the novelBrachypodium distachyon and Setaria italica promoters as compared to YFPexpression driven by the ZM Ubi1 and OS Act1 promoters.

FIG. 22 is a plasmid map showing the synthesized Panicum virgatumUbiquitin1 promoter genetic element and flanking seamless cloningoverhang location.

FIG. 23 is plasmid map showing the synthesized Panicum virgatumUbiquitin1 3′UTR genetic element and flanking seamless cloning overhanglocation.

FIG. 24 is plasmid map showing the synthesized Brachypodium distachyonUbiquitin1C 3′UTR genetic element and flanking seamless cloning overhanglocation.

FIG. 25 is plasmid map showing the synthesized Brachypodium distachyonUbiquitin1 3′UTR genetic element and flanking seamless cloning overhanglocation.

FIG. 26 is plasmid map showing the synthesized Setaria italicaubiquitin2 (SI-Ubi2) 3′UTR genetic element and flanking seamless cloningoverhang location.

FIG. 27 is plasmid map showing the expression vector containing Panicumvirgatum Ubiquitin1 promoter and 3′UTR fused to PhiYFP reporter gene.

FIG. 28 is plasmid map showing the expression vector containingBrachypodium distachyon Ubiquitin1 C promoter and 3′UTR fused to PhiYFPreporter gene.

FIG. 29 is a plasmid map showing the expression vector containingSetaria italica ubiquitin2 promoter and 3′UTR fused to PhiYFP reportergene.

FIG. 30 is plasmid map showing the expression vector containingBrachypodium distachyon Ubiquitin1 promoter and 3′UTR fused to PhiYFPreporter gene.

FIG. 31 is a plasmid map showing the binary expression vector containingBrachypodium distachyon Ubiquitin1 C promoter fused to yellowfluorescent protein (Phi YFP) marker gene coding region containingST-LS1 intron followed by fragment comprising a Brachypodium distachyonUbiquitin1 C 3′UTR.

FIG. 32 is a plasmid map showing the binary expression vector containingPanicum virgatum Ubiquitin1 promoter fused to yellow fluorescent protein(Phi YFP) marker gene coding region containing ST-LS1 intron followed byfragment comprising a Panicum virgatum Ubiquitin1 3′UTR.

FIG. 33 is a plasmid map showing the binary expression vector containingSetaria italica ubiquitin2 promoter fused to yellow fluorescent protein(Phi YFP) marker gene coding region containing ST-LS1 intron followed byfragment comprising a Setaria italica ubiquitin2 3′UTR.

FIG. 34 is a plasmid map showing the binary expression vector containingBrachypodium distachyon Ubiquitin1 promoter fused to yellow fluorescentprotein (Phi YFP) marker gene coding region containing ST-LS1 intronfollowed by fragment comprising a Brachypodium distachyon Ubiquitin13′UTR.

FIG. 35 presents the Brachypodium distachyon Ubiquitin1 C codingsequence and putative promoter (upstream sequence of ATG). The upstreampromoter sequence is underlined, the 5′-UTR sequence is presented inuppercase, the intron is boxed, the Ubi1 CDS is in italics, the 3′-UTRand the transcription termination sequence is downstream of TAA(Translational Stop Codon).

FIG. 36 presents the Brachypodium distachyon Ubiquitin 1 coding sequenceand putative promoter. The upstream promoter is underlined, the 5′UTRsequence is in uppercase, the intron is boxed, the CDS is in italics,the 3′-UTR and transcription termination sequence is downstream of TAA(Translational Stop Codon).

FIG. 37 presents the Setaria italica Ubiquitin2 coding sequence andputative promoter. The upstream promoter is underlined, the 5′UTRsequence is in uppercase, the intron is boxed, the CDS is in italics,the 3′-UTR and transcription termination sequence is downstream of TAA(Translational Stop Codon).

FIG. 38 presents the Panicum virgatum (Switchgrass) Ubiquitin 1 codingsequence and putative promoter. The upstream promoter is underlined, the5′UTR sequence is in uppercase, the intron is boxed, the CDS is initalics, the 3′-UTR and transcription termination sequence is downstreamof TAA (Translational Stop Codon).

DETAILED DESCRIPTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

As used herein, the term “backcrossing” refers to a process in which abreeder crosses hybrid progeny back to one of the parents, for example,a first generation hybrid F1 with one of the parental genotypes of theF1 hybrid.

A “promoter” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. A promoter may contain specific sequencesthat are recognized by transcription factors. These factors may bind toa promoter DNA sequence, which results in the recruitment of RNApolymerase. For purposes of defining the present invention, the promotersequence is bounded at its 3′ terminus by the transcription initiationsite and extends upstream (5′ direction) to include the minimum numberof bases or elements necessary to initiate transcription at levelsdetectable above background. Within the promoter sequence will be founda transcription initiation site (conveniently defined for example, bymapping with nuclease S1), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase. The promotermay be operatively associated with other expression control sequences,including enhancer and repressor sequences.

For the purposes of the present disclosure, a “gene,” includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

As used herein the terms “native” or “natural” define a condition foundin nature. A “native DNA sequence” is a DNA sequence present in naturethat was produced by natural means or traditional breeding techniquesbut not generated by genetic engineering (e.g., using molecularbiology/transformation techniques).

As used herein a “transgene” is defined to be a nucleic acid sequencethat encodes a gene product, including for example, but not limited to,an mRNA. In one embodiment the transgene is an exogenous nucleic acid,where the transgene sequence has been introduced into a host cell bygenetic engineering (or the progeny thereof) where the transgene is notnormally found. In one example, a transgene encodes an industrially orpharmaceutically useful compound, or a gene encoding a desirableagricultural trait (e.g., an herbicide-resistance gene). In yet anotherexample, a transgene is an antisense nucleic acid sequence, whereinexpression of the antisense nucleic acid sequence inhibits expression ofa target nucleic acid sequence. In one embodiment the transgene is anendogenous nucleic acid, wherein additional genomic copies of theendogenous nucleic acid are desired, or a nucleic acid that is in theantisense orientation with respect to the sequence of a target nucleicacid in a host organism.

As used herein the term “non-ubiquitin transgene” is any transgene thathas less than 80% sequence identity with the Zea may Ubiquitin 1 codingsequence (SEQ ID NO:27).

“Gene expression” as defined herein is the conversion of theinformation, contained in a gene, into a gene product.

A “gene product” as defined herein is any product produced by the gene.For example the gene product can be the direct transcriptional productof a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA,ribozyme, structural RNA or any other type of RNA) or a protein producedby translation of a mRNA. Gene products also include RNAs which aremodified, by processes such as capping, polyadenylation, methylation,and editing, and proteins modified by, for example, methylation,acetylation, phosphorylation, ubiquitination, ADP-ribosylation,myristilation, and glycosylation. Gene expression can be influenced byexternal signals, for example, exposure of a cell, tissue, or organismto an agent that increases or decreases gene expression. Expression of agene can also be regulated anywhere in the pathway from DNA to RNA toprotein. Regulation of gene expression occurs, for example, throughcontrols acting on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization, or degradationof specific protein molecules after they have been made, or bycombinations thereof. Gene expression can be measured at the RNA levelor the protein level by any method known in the art, including, withoutlimitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ,or in vivo protein activity assay(s).

As used herein, the term “intron” is defined as any nucleic acidsequence comprised in a gene (or expressed nucleotide sequence ofinterest) that is transcribed but not translated. Introns includeuntranslated nucleic acid sequence within an expressed sequence of DNA,as well as corresponding sequence in RNA molecules transcribedtherefrom. A construct described herein can also contain sequences thatenhance translation and/or mRNA stability such as introns. An example ofone such intron is the first intron of gene II of the histone H3 variantof Arabidopsis thaliana or any other commonly known intron sequence.Introns can be used in combination with a promoter sequence to enhancetranslation and/or mRNA stability.

As used herein, the terms “5′ untranslated region” or “5′-UTR” isdefined as the untranslated segment in the 5′ terminus of pre-mRNAs ormature mRNAs. For example, on mature mRNAs, a 5′-UTR typically harborson its 5′ end a 7-methylguanosine cap and is involved in many processessuch as splicing, polyadenylation, mRNA export towards the cytoplasm,identification of the 5′ end of the mRNA by the translational machinery,and protection of the mRNAs against degradation.

As used herein, the terms “transcription terminator” is defined as thetranscribed segment in the 3′ terminus of pre-mRNAs or mature mRNAs. Forexample, longer stretches of DNA beyond “polyadenylation signal” site istranscribed as a pre-mRNA. This DNA sequence usually contains one ormore transcription termination signals for the proper processing of thepre-mRNA into mature mRNA.

As used herein, the term “3′ untranslated region” or “3′-UTR” is definedas the untranslated segment in a 3′ terminus of the pre-mRNAs or maturemRNAs. For example, on mature mRNAs this region harbors the poly-(A)tail and is known to have many roles in mRNA stability, translationinitiation, and mRNA export.

As used herein, the term “polyadenylation signal” designates a nucleicacid sequence present in mRNA transcripts that allows for transcripts,when in the presence of a poly-(A) polymerase, to be polyadenylated onthe polyadenylation site, for example, located 10 to 30 bases downstreamof the poly-(A) signal. Many polyadenylation signals are known in theart and are useful for the present invention. An exemplary sequenceincludes AAUAAA and variants thereof, as described in Loke J., et al.,(2005) Plant Physiology 138(3); 1457-1468.

The term “isolated” as used herein means having been removed from itsnatural environment, or removed from other compounds present when thecompound is first formed. The term “isolated” embraces materialsisolated from natural sources as well as materials (e.g., nucleic acidsand proteins) recovered after preparation by recombinant expression in ahost cell, or chemically-synthesized compounds such as nucleic acidmolecules, proteins, and peptides.

The term “purified,” as used herein relates to the isolation of amolecule or compound in a form that is substantially free ofcontaminants normally associated with the molecule or compound in anative or natural environment, or substantially enriched inconcentration relative to other compounds present when the compound isfirst formed, and means having been increased in purity as a result ofbeing separated from other components of the original composition. Theterm “purified nucleic acid” is used herein to describe a nucleic acidsequence which has been separated, produced apart from, or purified awayfrom other biological compounds including, but not limited topolypeptides, lipids and carbohydrates, while effecting a chemical orfunctional change in the component (e.g., a nucleic acid may be purifiedfrom a chromosome by removing protein contaminants and breaking chemicalbonds connecting the nucleic acid to the remaining DNA in thechromosome).

As used herein, the terms “homology-based gene silencing” or “HBGS” aregeneric terms that include both transcriptional gene silencing andposttranscriptional gene silencing. Silencing of a target locus by anunlinked silencing locus can result from transcription inhibition(transcriptional gene silencing; TGS) or mRNA degradation(post-transcriptional gene silencing; PTGS), owing to the production ofdouble-stranded RNA (dsRNA) corresponding to promoter or transcribedsequences, respectively. Involvement of distinct cellular components ineach process suggests that dsRNA-induced TGS and PTGS likely result fromthe diversification of an ancient common mechanism. However, a strictcomparison of TGS and PTGS has been difficult to achieve because itgenerally relies on the analysis of distinct silencing loci. A singletransgene locus can be described to trigger both TGS and PTGS, owing tothe production of dsRNA corresponding to promoter and transcribedsequences of different target genes.

As used herein, the terms “nucleic acid molecule”, “nucleic acid”, or“polynucleotide” (all three terms are synonymous with one another) referto a polymeric form of nucleotides, which may include both sense andanti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms, andmixed polymers thereof. “A nucleotide” may refer to a ribonucleotide,deoxyribonucleotide, or a modified form of either type of nucleotide. Anucleic acid molecule is usually at least 10 bases in length, unlessotherwise specified. The terms may refer to a molecule of RNA or DNA ofindeterminate length. The terms include single- and double-strandedforms of DNA. A nucleic acid molecule may include either or bothnaturally-occurring and modified nucleotides linked together bynaturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be modified chemically or biochemically, ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications (e.g., uncharged linkages: for example, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.;charged linkages: for example, phosphorothioates, phosphorodithioates,etc.; pendent moieties: for example, peptides; intercalators: forexample, acridine, psoralen, etc.; chelators; alkylators; and modifiedlinkages: for example, alpha anomeric nucleic acids, etc.). The term“nucleic acid molecule” also includes any topological conformation,including single-stranded, double-stranded, partially duplexed,triplexed, hairpinned, circular, and padlocked conformations.

Transcription proceeds in a 5′ to 3′ manner along a DNA strand. Thismeans that RNA is made by sequential addition ofribonucleotide-5′-triphosphates to the 3′ terminus of the growing chain(with a requisite elimination of the pyrophosphate). In either a linearor circular nucleic acid molecule, discrete elements (e.g., particularnucleotide sequences) may be referred to as being “upstream” relative toa further element if they are bonded or would be bonded to the samenucleic acid in the 5′ direction from that element. Similarly, discreteelements may be “downstream” relative to a further element if they areor would be bonded to the same nucleic acid in the 3′ direction fromthat element.

As used herein, the term “base position,” refers to the location of agiven base or nucleotide residue within a designated nucleic acid. Adesignated nucleic acid may be defined by alignment with a referencenucleic acid.

As used herein, the term “hybridization” refers to a process whereoligonucleotides and their analogs hybridize by hydrogen bonding, whichincludes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary bases. Generally, nucleic acid molecules consistof nitrogenous bases that are either pyrimidines (cytosine (C), uracil(U), and thymine (T)) or purines (adenine (A) and guanine (G)). Thesenitrogenous bases form hydrogen bonds between a pyrimidine and a purine,and bonding of a pyrimidine to a purine is referred to as “basepairing.” More specifically, A will hydrogen bond to T or U, and G willbond to C. “Complementary” refers to the base pairing that occursbetween two distinct nucleic acid sequences or two distinct regions ofthe same nucleic acid sequence.

As used herein, the terms “specifically hybridizable” and “specificallycomplementary” refers to a sufficient degree of complementarity suchthat stable and specific binding occurs between an oligonucleotide andthe DNA or RNA target. Oligonucleotides need not be 100% complementaryto its target sequence to specifically hybridize. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget DNA or RNA molecule interferes with the normal function of thetarget DNA or RNA, and there is sufficient degree of complementarity toavoid non-specific binding of an oligonucleotide to non-target sequencesunder conditions where specific binding is desired, for example underphysiological conditions in the case of in vivo assays or systems. Suchbinding is referred to as specific hybridization. Hybridizationconditions resulting in particular degrees of stringency will varydepending upon the nature of the chosen hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength(especially Na⁺ and/or Mg²⁺ concentration) of a hybridization bufferwill contribute to the stringency of hybridization, though wash timesalso influence stringency. Calculations regarding hybridizationconditions required for attaining particular degrees of stringency arediscussed in Sambrook et al. (ed.), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, chs. 9 and 11.

As used herein, the term “stringent conditions” encompasses conditionsunder which hybridization will only occur if there is less than 50%mismatch between the hybridization molecule and the DNA target.“Stringent conditions” include further particular levels of stringency.Thus, as used herein, “moderate stringency” conditions are those underwhich molecules with more than 50% sequence mismatch will not hybridize;conditions of “high stringency” are those under which sequences withmore than 20% mismatch will not hybridize; and conditions of “very highstringency” are those under which sequences with more than 10% mismatchwill not hybridize. In particular embodiments, stringent conditions caninclude hybridization at 65° C., followed by washes at 65° C. with0.1×SSC/0.1% SDS for 40 minutes. The following are representative,non-limiting hybridization conditions:

-   -   Very High Stringency: hybridization in 5×SSC buffer at 65° C.        for 16 hours; wash twice in 2×SSC buffer at room temperature for        15 minutes each; and wash twice in 0.5×SSC buffer at 65° C. for        20 minutes each.    -   High Stringency: Hybridization in 5-6×SSC buffer at 65-70° C.        for 16-20 hours; wash twice in 2×SSC buffer at room temperature        for 5-20 minutes each; and wash twice in 1×SSC buffer at        55-70° C. for 30 minutes each.    -   Moderate Stringency: Hybridization in 6×SSC buffer at room        temperature to 55° C. for 16-20 hours; wash at least twice in        2×-3×SSC buffer at room temperature to 55° C. for 20-30 minutes        each.        In an embodiment, specifically hybridizable nucleic acid        molecules can remain bound under very high stringency        hybridization conditions. In an embodiment, specifically        hybridizable nucleic acid molecules can remain bound under high        stringency hybridization conditions. In an embodiment,        specifically hybridizable nucleic acid molecules can remain        bound under moderate stringency hybridization conditions.

As used herein, the term “oligonucleotide” refers to a short nucleicacid polymer. Oligonucleotides may be formed by cleavage of longernucleic acid segments, or by polymerizing individual nucleotideprecursors. Automated synthesizers allow the synthesis ofoligonucleotides up to several hundred base pairs in length. Becauseoligonucleotides may bind to a complementary nucleotide sequence, theymay be used as probes for detecting DNA or RNA. Oligonucleotidescomposed of DNA (oligodeoxyribonucleotides) may be used in PCR, atechnique for the amplification of small DNA sequences. In PCR, anoligonucleotide is typically referred to as a “primer,” which allows aDNA polymerase to extend the oligonucleotide and replicate thecomplementary strand.

As used herein, the terms “Polymerase chain reaction” or “PCR” define aprocedure or technique in which minute amounts of nucleic acid, RNAand/or DNA, are amplified as described in U.S. Pat. No. 4,683,195 issuedJul. 28, 1987. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers may coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (StocktonPress, NY, 1989).

As used herein, the term “primer” refers to an oligonucleotide capableof acting as a point of initiation of synthesis along a complementarystrand when conditions are suitable for synthesis of a primer extensionproduct. The synthesizing conditions include the presence of fourdifferent deoxyribonucleotide triphosphates and at least onepolymerization-inducing agent such as reverse transcriptase or DNApolymerase. These are present in a suitable buffer, which may includeconstituents which are co-factors or which affect conditions such as pHand the like at various suitable temperatures. A primer is preferably asingle strand sequence, such that amplification efficiency is optimized,but double stranded sequences can be utilized.

As used herein, the term “probe” refers to an oligonucleotide thathybridizes to a target sequence. In the TaqMan® or TaqMan®-style assayprocedure, the probe hybridizes to a portion of the target situatedbetween the annealing site of the two primers. A probe includes abouteight nucleotides, about ten nucleotides, about fifteen nucleotides,about twenty nucleotides, about thirty nucleotides, about fortynucleotides, or about fifty nucleotides. In some embodiments, a probeincludes from about eight nucleotides to about fifteen nucleotides. Aprobe can further include a detectable label, e.g., a fluorophore(TexasRed®, Fluorescein isothiocyanate, etc.,). The detectable label canbe covalently attached directly to the probe oligonucleotide, e.g.,located at the probe's 5′ end or at the probe's 3′ end. A probeincluding a fluorophore may also further include a quencher, e.g., BlackHole Quencher™, Iowa Black™, etc.

As used herein, the terms “sequence identity” or “identity” can be usedinterchangeably and refer to nucleic acid residues in two sequences thatare the same when aligned for maximum correspondence over a specifiedcomparison window.

As used herein, the term “percentage of sequence identity” refers to avalue determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences or amino acid sequences) over a comparisonwindow, wherein the portion of a sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to a referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. A percentage is calculated bydetermining the number of positions at which an identical nucleic acidor amino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity. Methods foraligning sequences for comparison are well known. Various programs andalignment algorithms are described in, for example: Smith and Waterman(1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol.48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444;Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang etal. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) MethodsMol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett.174:247-50.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™; Altschul et al. (1990) J. Mol. Biol.215:403-10) is available from several sources, including the NationalCenter for Biotechnology Information (Bethesda, Md.), and on theinternet, for use in connection with several sequence analysis programs.A description of how to determine sequence identity using this programis available on the internet under the “help” section for BLAST™. Forcomparisons of nucleic acid sequences, the “Blast 2 sequences” functionof the BLAST™ (Blastn) program may be employed using the defaultparameters. Nucleic acid sequences with even greater similarity to thereference sequences will show increasing percentage identity whenassessed by this method.

As used herein, the term “operably linked” refers to two components thathave been placed into a functional relationship with one another. Theterm, “operably linked,” when used in reference to a regulatory sequenceand a coding sequence, means that the regulatory sequence affects theexpression of the linked coding sequence. “Regulatory sequences,”“regulatory elements”, or “control elements,” refer to nucleic acidsequences that influence the timing and level/amount of transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters; translation leadersequences; 5′ and 3′ untranslated regions, introns; enhancers; stem-loopstructures; repressor binding sequences; termination sequences;polyadenylation recognition sequences; etc. Particular regulatorysequences may be located upstream and/or downstream of a coding sequenceoperably linked thereto. Also, particular regulatory sequences operablylinked to a coding sequence may be located on the associatedcomplementary strand of a double-stranded nucleic acid molecule. Linkingcan be accomplished by ligation at convenient restriction sites. If suchsites do not exist, synthetic oligonucleotide adaptors or linkers areused in accordance with conventional practice. However, elements neednot be contiguous to be operably linked.

As used herein, the term “transformation” encompasses all techniques bywhich a nucleic acid molecule can be introduced into such a cell.Examples include, but are not limited to: transfection with viralvectors; transformation with plasmid vectors; electroporation;lipofection; microinjection (Mueller et al. (1978) Cell 15:579-85);Agrobacterium-mediated transfer; direct DNA uptake; whiskers-mediatedtransformation; and microprojectile bombardment.

As used herein, the term “transduce” refers to a process where a virustransfers nucleic acid into a cell.

The terms “polylinker” or “multiple cloning site” as used herein definesa cluster of three or more Type −2 restriction enzyme sites locatedwithin 10 nucleotides of one another on a nucleic acid sequence.Constructs comprising a polylinker are utilized for the insertion and/orexcision of nucleic acid sequences such as the coding region of a gene.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence. Type −2 restrictionenzymes recognize and cleave DNA at the same site, and include but arenot limited to XbaI, BamHI, HindIII, EcoRI, XhoI, SalI, KpnI, AvaI, PstIand SmaI.

The term “vector” is used interchangeably with the terms “construct”,“cloning vector” and “expression vector” and means the vehicle by whicha DNA or RNA sequence (e.g. a foreign gene) can be introduced into ahost cell, so as to transform the host and promote expression (e.g.transcription and translation) of the introduced sequence. A “non-viralvector” is intended to mean any vector that does not comprise a virus orretrovirus. In some embodiments a “vector” is a sequence of DNAcomprising at least one origin of DNA replication and at least oneselectable marker gene. Examples include, but are not limited to, aplasmid, cosmid, bacteriophage, bacterial artificial chromosome (BAC),or virus that carries exogenous DNA into a cell. A vector can alsoinclude one or more genes, antisense molecules, and/or selectable markergenes and other genetic elements known in the art. A vector maytransduce, transform, or infect a cell, thereby causing the cell toexpress the nucleic acid molecules and/or proteins encoded by thevector.

The term “plasmid” defines a circular strand of nucleic acid capable ofautosomal replication in either a prokaryotic or a eukaryotic host cell.The term includes nucleic acid which may be either DNA or RNA and may besingle- or double-stranded. The plasmid of the definition may alsoinclude the sequences which correspond to a bacterial origin ofreplication.

The term “selectable marker gene” as used herein defines a gene or otherexpression cassette which encodes a protein which facilitatesidentification of cells into which the selectable marker gene isinserted. For example a “selectable marker gene” encompasses reportergenes as well as genes used in plant transformation to, for example,protect plant cells from a selective agent or provideresistance/tolerance to a selective agent. In one embodiment only thosecells or plants that receive a functional selectable marker are capableof dividing or growing under conditions having a selective agent.Examples of selective agents can include, for example, antibiotics,including spectinomycin, neomycin, kanamycin, paromomycin, gentamicin,and hygromycin. These selectable markers include neomycinphosphotransferase (npt II), which expresses an enzyme conferringresistance to the antibiotic kanamycin, and genes for the relatedantibiotics neomycin, paromomycin, gentamicin, and G418, or the gene forhygromycin phosphotransferase (hpt), which expresses an enzymeconferring resistance to hygromycin. Other selectable marker genes caninclude genes encoding herbicide resistance including bar or pat(resistance against glufosinate ammonium or phosphinothricin),acetolactate synthase (ALS, resistance against inhibitors such assulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs),pyrimidinyl oxybenzoates (POBs), and sulfonylamino carbonyltriazolinones that prevent the first step in the synthesis of thebranched-chain amino acids), glyphosate, 2,4-D, and metal resistance orsensitivity. Examples of “reporter genes” that can be used as aselectable marker gene include the visual observation of expressedreporter gene proteins such as proteins encoding β-glucuronidase (GUS),luciferase, green fluorescent protein (GFP), yellow fluorescent protein(YFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT),alkaline phosphatase, and the like. The phrase “marker-positive” refersto plants that have been transformed to include a selectable markergene.

As used herein, the term “detectable marker” refers to a label capableof detection, such as, for example, a radioisotope, fluorescentcompound, bioluminescent compound, a chemiluminescent compound, metalchelator, or enzyme. Examples of detectable markers include, but are notlimited to, the following: fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In anembodiment, a detectable marker can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

As used herein, the term “detecting” is used in the broadest sense toinclude both qualitative and quantitative measurements of a specificmolecule, for example, measurements of a specific polypeptide.

As used herein, the terms “cassette”, “expression cassette” and “geneexpression cassette” refer to a segment of DNA that can be inserted intoa nucleic acid or polynucleotide at specific restriction sites or byhomologous recombination. As used herein the segment of DNA comprises apolynucleotide that encodes a polypeptide of interest, and the cassetteand restriction sites are designed to ensure insertion of the cassettein the proper reading frame for transcription and translation. In anembodiment, an expression cassette can include a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.In an embodiment, a gene expression cassette may also include elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

As used herein a “linker” or “spacer” is a bond, molecule or group ofmolecules that binds two separate entities to one another. Linkers andspacers may provide for optimal spacing of the two entities or mayfurther supply a labile linkage that allows the two entities to beseparated from each other. Labile linkages include photocleavablegroups, acid-labile moieties, base-labile moieties and enzyme-cleavablegroups.

As used herein, the term “control” refers to a sample used in ananalytical procedure for comparison purposes. A control can be“positive” or “negative”. For example, where the purpose of ananalytical procedure is to detect a differentially expressed transcriptor polypeptide in cells or tissue, it is generally preferable to includea positive control, such as a sample from a known plant exhibiting thedesired expression, and a negative control, such as a sample from aknown plant lacking the desired expression.

As used herein, the term “plant” includes a whole plant and anydescendant, cell, tissue, or part of a plant. A class of plant that canbe used in the present invention is generally as broad as the class ofhigher and lower plants amenable to mutagenesis including angiosperms(monocotyledonous and dicotyledonous plants), gymnosperms, ferns andmulticellular algae. Thus, “plant” includes dicot and monocot plants.The term “plant parts” include any part(s) of a plant, including, forexample and without limitation: seed (including mature seed and immatureseed); a plant cutting; a plant cell; a plant cell culture; a plantorgan (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots,stems, and explants). A plant tissue or plant organ may be a seed,protoplast, callus, or any other group of plant cells that is organizedinto a structural or functional unit. A plant cell or tissue culture maybe capable of regenerating a plant having the physiological andmorphological characteristics of the plant from which the cell or tissuewas obtained, and of regenerating a plant having substantially the samegenotype as the plant. In contrast, some plant cells are not capable ofbeing regenerated to produce plants. Regenerable cells in a plant cellor tissue culture may be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks, or stalks.

Plant parts include harvestable parts and parts useful for propagationof progeny plants. Plant parts useful for propagation include, forexample and without limitation: seed; fruit; a cutting; a seedling; atuber; and a rootstock. A harvestable part of a plant may be any usefulpart of a plant, including, for example and without limitation: flower;pollen; seedling; tuber; leaf; stem; fruit; seed; and root.

A plant cell is the structural and physiological unit of the plant,comprising a protoplast and a cell wall. A plant cell may be in the formof an isolated single cell, or an aggregate of cells (e.g., a friablecallus and a cultured cell), and may be part of a higher organized unit(e.g., a plant tissue, plant organ, and plant). Thus, a plant cell maybe a protoplast, a gamete producing cell, or a cell or collection ofcells that can regenerate into a whole plant. As such, a seed, whichcomprises multiple plant cells and is capable of regenerating into awhole plant, is considered a “plant cell” in embodiments herein.

The term “protoplast,” as used herein, refers to a plant cell that hadits cell wall completely or partially removed, with the lipid bilayermembrane thereof naked, and thus includes protoplasts, which have theircell wall entirely removed, and spheroplasts, which have their cell wallonly partially removed, but is not limited thereto. Typically, aprotoplast is an isolated plant cell without cell walls which has thepotency for regeneration into cell culture or a whole plant.

Unless otherwise specifically explained, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this disclosure belongs.Definitions of common terms in molecular biology can be found in, forexample: Lewin, Genes V, Oxford University Press, 1994 (ISBN0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers(ed.), Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

EMBODIMENTS

As disclosed herein novel recombinant constructs are provided forexpressing a non-ubiquitin transgene using the regulatory sequences of aubiquitin gene from Panicum virgatum, Brachypodium distachyon, orSetaria italica. These constructs can be used to transform cells,including plant cells, to produce complete organisms that express thetransgene gene product in their cells.

Regulatory Elements

Plant promoters used for basic research or biotechnological applicationare generally unidirectional, directing only one gene that has beenfused at its 3′ end (downstream). It is often necessary to introducemultiple genes into plants for metabolic engineering and trait stackingand therefore, multiple promoters are typically required in transgeniccrops to drive the expression of multiple genes.

Development of transgenic products is becoming increasingly complex,which requires stacking multiple transgenes into a single locus.Traditionally, each transgene usually requires a promoter for expressionwherein multiple promoters are required to express different transgeneswithin one gene stack. This frequently leads to repetitive use of thesame promoter within one transgene stack to obtain similar levels ofexpression patterns of different transgenes for expression of a singlepolygenic trait. Multi-gene constructs driven by the same promoter areknown to cause gene silencing resulting in less efficacious transgenicproducts in the field. Excess of transcription factor (TF)-binding sitesdue to promoter repetition can cause depletion of endogenous TFs leadingto transcriptional inactivation. The silencing of transgenes will likelyundesirably affect performance of a transgenic plant produced to expresstransgenes. Repetitive sequences within a transgene may lead to geneintra locus homologous recombination resulting in polynucleotiderearrangements.

It is desirable to use diversified promoters for the expression ofdifferent transgenes in a gene stack. In an embodiment, diversifiedconstitutive ubiquitin obtained from different plant species can drivetranscription of multiple transcription units, including RNAi,artificial miRNA, or hairpin-loop RNA sequences.

Provided are methods and constructs using a constitutive ubiquitin(Ubi1) promoter to express non-ubiquitin transgenes in plant. In anembodiment, a promoter can be the Brachypodium distachyon ubiquitin1 C(Ubi1C) promoter.

(SEQ ID NO: 1) CTGCTCGTTCAGCCCACAGTAACACGCCGTGCGACATGCAGATGCCCTCCACCACGCCGACCAACCCCAAGTCCGCCGCGCTCGTCCACGGCGCCATCCGCATCCGCGCGTCAACGTCATCCGGAGGAGGCGAGCGCGATGTCGACGGCCACGGCGGCGGCGGACACGACGGCGACGCCCCGACTCCGCGCGCGCGTCAAGGCTGCAGTGGCGTCGTGGTGGCCGTCCGCCTGCACGAGATCCCCGCGTGGACGAGCGCCGCCTCCACCCAGCCCCTATATCGAGAAATCAACGGTGGGCTCGAGCTCCTCAGCAACCTCCCCACCCCCCCTTCCGACCACGCTCCCTTCCCCCGTGCCCCTCTTCTCCGTAAACCCGAGCCGCCGAGAACAACACCAACGAAAGGGCGAAGAGAATCGCCATAGAGAGGAGATGGGCGGAGGCGGATAGTTTCAGCCATTCACGGAGAAATGGGGAGGAGAGAACACGACATCATACGGACGCGACCCTCTAGCTGGCTGGCTGTCCTAAAGAATCGAACGGAATCGCTGCGCCAGGAGAAAACGAACGGTCCTGAAGCATGTGCGCCCGGTTCTTCCAAAACACTTATCTTTAAGATTGAAGTAGTATATATGACTGAAATTTTTACAAGGTTTTTCCCCATAAAACAGGTGAGCTTATCTCATCCTTTTGTTTAGGATGTACGTATTATATATGACTGAATATTTTTTATTTTCATTGAATGAAGATTTTCGACCCCCCAAAAATAAAAAACGGAGGGAGTACCTTTGTGCCGTGTATATGGACTAGAGCCATCGGGACGTTTCCGGAGACTGCGTGGTGGGGGCGATGGACGCACAACGACCGCATTTTCGGTTGCCGACTCGCCGTTCGCATCTGGTAGGCACGACTCGTCGGGTTCGGCTCTTGCGTGAGCCGTGACGTAACAGACCCGTTCTCTTCCCCCGTCTGGCCATCCATAAATCCCCCCTCCATCGGCTTCCCTTTCCTCAATCCAGCACCCTGATTIn an embodiment, a promoter can be the Brachypodium distachyonubiquitin 1 (Ubi1) promoter.

(SEQ ID NO: 2) GGCGTCAGGACTGGCGAAGTCTGGACTCTGCAGGGCCGAACTGCTGAAGACGAAGCAGAGGAAGAGAAAGGGAAGTGTTCGACTTGTAATTTGTAGGGGTTTTTTTTAGAGGAACTTGTAATTTGTAGGTGGGCTGGCCTCGTTGGAAAAACGATGCTGGCTGGTTGGGCTGGGCCGATGTACGCTTGCAAACAACTTGTGGCGGCCCGTTCTGGACGAGCAGGAGTTTCTTTTTTGTTCTCACTTTTCTGGTCTTCTTTAGTTACGGAGTACCTTTTGTTTTTTAAAGGAGTTACCTTTTTTTTAGGAATTCTTTAGTTACCTTTCGCTTGCTCTCAAAAAATATTTAACTTTCGCTTTTTTTCATTTTAATTTTTGCAACTATTTACGAGTTTCATGAATGCTTATTTTCCAGCATATCATTATTTGCAAGTATTTTTATGCCGTATGTATTGGACGAGAGCCATCGGGACTGTTCCAGAGACTGCGTGGTGGGGACGGCTCCCAACCGCCTTTTCTATCTCTGTTCGCATCCGGTGGCCGACTTGGCTCGCGCGTGAGCCGTGACGTAACAGACTTGGTCTCTTCCCCATCTGGCCATCTATAAATTCCCCCATCGATC GACCCTCCCTTTCCIn an embodiment, a promoter can be the Setaria italica ubiquitin 2(Ubi2) promoter.

(SEQ ID NO: 3) TGCGTCTGGACGCACAAGTCATAGCATTATCGGCTAAAATTTCTTAATTTCTAAATTAGTCATATCGGCTAAGAAAGTGGGGAGCACTATCATTTCGTAGAACAAGAACAAGGTATCATATATATATATATATATAATATTTAAACTTTGTTAAGTGGAATCAAAGTGCTAGTATTAATGGAGTTTCATGTGCATTAAATTTTATGTCACATCAGCAATTTTGTTGACTTGGCAAGGTCATTTAGGGTGTGTTTGGAAGACAGGGGCTATTAGGAGTATTAAACATAGTCTAATTACAAAACTAATTGCACAACCGCTAAGCTGAATCGCGAGATGGATCTATTAAGCTTAATTAGTCCATGATTTGACAATGTGGTGCTACAATAACCATTTGCTAATGATGGATTACTTAGGTTTAATAGATTCGTCTCGTGATTTAGCCTATGGGTTCTGCTATTAATTTTGTAATTAGCTCATATTTAGTTCTTATAATTAGTATCCGAACATCCAATGTGACATGCTAAAGTTTAACCCTGGTATCCAAATGAAGTCTTATGAGAGTTTCATCACTCCGGTGGTATATGTACTTAGGCTCCGTTTTCTTCCACCGACTTATTTTTAGCACCCGTCACATTGAATGTTTAGATACTAATTAGAAGTATTAAACGTAGACTATTTACAAAATCCATTACATAAGACGAATCTAAACGGCGAGACGAATCTATTAAACCTAATTAGTCCATGATTTGACAATGTGTTGCTACAGTAAACATTTGCTAATGATGGATTAATTAGGCTTAATAGATTCGTCTCGCCGTTTAGCCTCCACTTATGTAATGGGTTTTCTAAACAATCTACGTTTAATACTCCTAATTAGTATCTAAATATTCAATGTGACACGTGCTAAAAATAAGTCAGTGGAAGGAAGAGAACGTCCCCTTAGTTTTCCATCTTATTAATTGTACGATGAAACTGTGCAGCCAGATGATTGACAATCGCAATACTTCAACTAGTGGGCCATGCACATCAGCGACGTGTAACGTCGTGA GTTGCTGTTCCCGTAGIn an embodiment, a promoter can be the Panicum virgatum (Switchgrass)ubiquitin 1 promoter.

(SEQ ID NO: 35) TTGAATTTTAATTTCAAATTTTGCAGGGTAGTAGTGGACATCACAATACATATTTAGAAAAAGTTTTATAATTTTCCTCCGTTAGTTTTCATATAATTTTGAACTCCAACGATTAATCTATTATTAAATATCCCGATCTATCAAAATAATGATAAAAATTTATGATTAATTTTTCTAACATGTGTTATGGTGTGTACTATCGTCTTATAAAATTTCAACTTAAAACTCCACCTATACATGGAGAAATGAAAAAGACGAATTACAGTAGGGAGTAATTTGAACCAAATGGAATAGTTTGAGGGTAAAATGAACTAAACAATAGTTTAGGAGGTTATTCAGATTTTAGTTATAGTTGAGAGGAGTAATTTAGACTTTTTCCTATCTTGAATTGTTGACGGCTCTCCTATCGGATATCGGATGGAGTCTTTCAGCCCAACATAACTTCATTCGGGCCCAAACGTTCGTCCATCCAGCCTAGGGAGAACATTTTGCCCATGATATCTGTTTTTCTTTTTTTCTATTTTCACTGGTATTATAGGAGGGAAATATACAACGTGTTCACCTTTGGTTTCATTCTTGTTCCATCTGAATTTATCTAAAACTGTGTTTGAACTTCGTAAGAATTTTGTTCGATCTGTCCGGTACATCGTGTTGATAGGTGGCCTCCGAGATTCTTCTTTTTAACCGGCAAAGTAAAATAATCTCAGCTCCAGCCTAACGTCAATTATCAGAGAGAGAAAAAAATATTTTTTTATGATTGATCGGAAACCAACCGCCTTACGTGTCGATCCTGGTTCCTGGCCGGCACGGCGGAGGAAAGCGACCGACCTCGCAACGCCGGCGCACGGCGCCGCCGTGTTGGACTTGGTCTCCCGCGACTCCGTGGGCCTCGGCTTATCGCCGCCGCTCCATCTCAACCGTCCGCTTGGACACGTGGAAGTTGATCCGTCGCGCACCAGCCTCGGAGGTAACCTAACTGCCCGTACTATAAATCCGGGATC CGGCCTCTCCAATCCCCATCGCCA

In an embodiment, a nucleic acid construct is provided comprising aubiquitin promoter. In an embodiment, the ubiquitin promoter is aPanicum virgatum, Brachypodium distachyon or Setaria italica ubiquitinpromoter. In an embodiment, a nucleic acid construct is providedcomprising a promoter, wherein the promoter is at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100%identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35. Inan embodiment, a nucleic acid construct is provided comprising aubiquitin promoter that is operably linked to a polylinker. In anembodiment, a gene expression cassette is provided comprising aubiquitin promoter that is operably linked to a non-ubiquitin transgene.In one embodiment the promoter consists of SEQ ID NO: 1, SEQ ID NO:2,SEQ ID NO:3, or SEQ ID NO:35. In an illustrative embodiment, a geneexpression cassette comprises a ubiquitin promoter that is operablylinked to the 5′ end of a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water us efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof.

In addition to a promoter, a 3′-untranslated gene region (i.e., 3′UTR)or terminator is needed for transcription termination andpolyadenylation of the mRNA. Proper transcription termination andpolyadenylation of mRNA is important for stable expression of transgene.The transcription termination becomes more critical for multigene stacksto avoid transcription read-through into next transgene. Similarly,non-polyadenylated aberrant RNA (aRNA) is a substrate for plantRNA-dependent RNA polymerases (RdRPs) to convert aRNA into doublestranded RNA (dsRNA) leading to small RNA production and transgenesilencing. Strong transcription terminators therefore are very usefulboth for single gene and multiple gene stacks. While a promoter isnecessary to drive transcription, a 3′-UTR gene region can terminatetranscription and initiate polyadenylation of a resulting mRNAtranscript for translation and protein synthesis. A 3′-UTR gene regionaids stable expression of a transgene.

In accordance with one embodiment a nucleic acid construct is providedcomprising a ubiquitin transcription terminator. In an embodiment, theubiquitin transcription terminator is a Panicum virgatum, Brachypodiumdistachyon or Setaria italica ubiquitin transcription terminator. In anembodiment, a nucleic acid construct is provided comprising atranscription terminator, wherein the transcription terminator is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, orSEQ ID NO:36. In an embodiment, a nucleic acid construct is providedcomprising a ubiquitin transcription terminator that is operably linkedto a polylinker. In an embodiment, a gene expression cassette isprovided comprising a ubiquitin transcription terminator that isoperably linked to the 3′ end of a non-ubiquitin transgene. In oneembodiment the transcription terminator consists of SEQ ID NO: 4, SEQ IDNO:5, SEQ ID NO:6, or SEQ ID NO:36. In an illustrative embodiment, agene expression cassette comprises a ubiquitin transcription terminatorthat is operably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water us efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof. In one embodiment a nucleicacid vector is provided comprising a transcription terminator operablylinked to either a polylinker sequence, a non-ubiquitin transgene or acombination of both, wherein the transcription terminator comprises SEQID NO: 4 or a sequence that has 90% sequence identity with SEQ ID NO: 4.In one embodiment the transcription terminator is less than 1 kb inlength, and in a further embodiment the transcription terminatorconsists of the 3′UTR sequence of SEQ ID NO: 4.

In an embodiment, a nucleic acid construct is provided comprising aubiquitin promoter as described herein and a 3′-UTR. In an embodiment,the nucleic acid construct comprises a ubiquitin 3′-UTR. In anembodiment, the ubiquitin 3′-UTR is a Panicum virgatum, Brachypodiumdistachyon or Setaria italica ubiquitin 3′-UTR. In an embodiment, a3′-UTR can be the Brachypodium distachyon ubiquitin1 C (Ubi1C) 3′-UTR.

(SEQ ID NO: 4) GTTTGTCAAAAACTGGCCTACAGTCTGCTGCCCCTGTTGGTCTGCCCCTTGGAAGTAGTCGTGTCTATGGTTATGTGAGAAGTCGTTGTGTTCTTTCTAATCCCGTACTGTTTGTGTGAACATCTGCTGCTGTCGTATTGCATCGTGAAGAATCCTGTTATGAATAAGTGAACATGAACCTTGTTCTGTGATTACGGCTTCGTGGTTATGCGAACGTTCTTACAAACGCAATTGCACCTGATGTAAAATCGTTTTTGCTAGCTGTATGGAACAAGTGCTCATGATGTTCATGCAAGATGCAATTCCAGCTTTTGTTGGTTTGTCATCTTTGTACTGTGCTTACCGCACATAAAGATTGCATCTTGCTTATTGCTTTGTTGCTTTGGTGCTCGTCCGCTTCTCCTTGCACCTTATCAAACCTTTGTTTAGATTCTCTTCTTATAGCACTTGGTAACTCTCAGCTTTACAACGCCAGTACTGTTTCTGAAATTTCATGACTGATAAAGCTGATAGATGGAGTACTAATATATGACATCTTTCCATAAATGTTCGGGTGCAGAGATATGGAGGCCCCAGGATCCTATTTACAGGATGAACCTACCTGGGCCGCTGTACGCATGACATCCGCGAGCAAGTCTGAGGTTCTCAATGTACACATGAAATTGATTTTTGCTGCGTTTGGCTTGGCTGATCGTTGCATTTGTTCTGATTCATCAGAGTTAAATAACGGATATATCAGCAAATATCCGCAGCATCCACACCGACCACACGTCCGGTTAACAGAGTCCCCCTGCCTTGCTTTAATTATTACGGAGTACTCCGCTATTAATCCTTAGATATGTTTCGAAGGAACTCAAACCTTCCTCCATCTGCAAATCTCAGTGCTTCAAAACTGGAATTAGATAATTGAAACCTTCATTCGGTTGCAATTCACAACTGCAAATTGAACAGCACTGTCAATTTCAATTTCGGGTTCACGATTCCACCGATAGGTTGACATGATC CATGATCCACCCATTGTACAACIn an embodiment, a 3′-UTR can be the Brachypodium distachyon ubiquitin1 (Ubi1) 3′-UTR.

(SEQ ID NO: 5) GCTTCTGCCGAACTGGTTCACAGTCTGCTGCCCTTGGTGGTCTGCCCCTTAGTGGTCATGCCTTTTGTTATGTGTCTTGCGTCCCAATCCTGTATCGTTTGTGTGAACATCTCTGCTGCTGTATAGCAGCTTGAATCCTGTTATGAATTTGTGAACCTGAACCTTGTTCCGTGAATCATGTTATGAATAAGTGAACCTGAACCTTGTTCCGTGATTATTGTTACAATCTGTTGTGCCGTATGGTTGGTCGTGTGTGATTTATGTTGAACTGGAGAACCAAGTTCGTTCCAGGACATATTGCAACCTAAGCTAAACCATGTAGAACTACTTGTTCTGGGAGACATAAAACGTCATTTTTATGCATTCGTAACATTTAAGCATACTACAATAATTGTATTGTCCTTTTCCTACTCATCCTTGAAACCATATGCCTCTTCTCAGCGCCTCTACATGCAGTGTGCTCAGAACAAACAGGCCCTGCCAGCTGCTTTTCAATTTTCCAATTAATAACCACAATAGTCGGACTATGGCATCTGTGGGTGACTATGCAAGATGTTGCTGTCAGGTCTCTGAAACTTTTCCCATGTATCTGTTGAAATTACCCAGTAAATTCATGCCTCTATTTAATCTGGCATGGTTGATTTTCAAACAGAATGTGTTTTTTTTTGTTCTGGAAGCTATATTGGTAAATAAATACAAAGCTGGAGTGTGATTATATTTCCAACAGATATTCAAGAAAATCTCAGTTGATTTATTTACTACTGTAGTATATATATATATCTTACAGTTGACTTCTCATATTTCAAACGACATGTGAGCACATTGTTCAGTTTCTTAGGATGTGTTGTGTGCTCAAAGGTGTAATTTTGCATTCTGCCCTCCGAGTAAACACTACACGTATTTTTTTGAGTGGCAGTGCATTTGATTACAAGGCAACAACAACAAAAACCTATGGCAAGATATCCTTCTTAGAGGCTGCCAGGATCATTTTGACTGAACTATGTA AGGCTGAAGAAAAGGIn an embodiment, a 3′-UTR can be the Setaria italica ubiquitin 2 (Ubi2)3′-UTR.

(SEQ ID NO: 6) GCCCATCGGTCATGGATGCTTCTACTGTACCTGGGTCGTCTGGTCTCTGCCTGTGTCACCTTTGAAGTACCTGTGTCGGGATTGTGTTTGGTCATGAACTGCAGTTTGTCTTTGATGTTCTTTTGTCTGGTCTTATGAACTGGTTGTATCTGTATGTTTACTGTAAACTGTTGTTGCGGTGCAGCAGTATGGCATCCGAATGAATAAATGATGTTTGGACTTAAATCTGTACTCTGTTTGTTTTCGGTTATGCCAGTTCTATATTGCCTGAGATCAGAATGTTTAGCTTTTGAGTTCTGTTTGGCTTGTGGTCGACTCCTGTTTCTTACTTGAGGCGTAACTCTGTTCTGGCAAACTCAAATGTCTAACTGAATGTTTTAGGACTTAATTGTTGGACAGATTAACGTGTTTGGTTTGTTTCTAGATTGTGATTCGGAAGGCTTGTTAGTTGTGGAATCAAGGAGAGCAGCTAGGTCTGTGCAGAACGTTATTTTGGATTTAAGCCTTCTCAGATTATGCCATTACTCTAAACCTAATGATATCATATTTCACTCGGGGATGTTGGAGTAGTCTTTTCTTTCTCCTGCAGACAAAATGATTTTGCTTTCGTGTGTGTACATGATTTTGTGCAACTGTTGCAACAACTGAAGTAGACAAGTTTTGACCTCACCAGAAGAATGAAAAAGATTTTGGAATTTGTTACATCGACAAACCATTGTAACTTGGCCCATCAGAATGCACAGAAGAGCGGCTACAAATTGACATGCGTTGCAAACTTTGCAATAGTTGATGCACATGTTTGCCATTGCCTGCCAGTCTTAGGAAAAGTGTGTGGTTCGAGAAATCTAAGCATATGTGCTCTGCTCACATTGCGTGGAACCCACACAGCTTTGTCACACTCTTGTCCACTCCAGAAGTCATTCCTGGCGCTGTTTACCCCTGGTAAAAGGTAACCGAAAACTTCTCAAGGCTGTACCCAAAACTGGAAGGAAATTTGGAGGAAATCTTTGCTTTTGATCGGCTCACTCTTTCIn an embodiment, a 3′-UTR can be the Panicum virgatum (Switchgrass)ubiquitin 1 3′-UTR.

(SEQ ID NO: 36) GCCTAGTGCTCCTGAGTTGCCTTTTGTCGTTATGGTCAACCTCTGGTTTAAGTCGTGTGAACTCTCTGCATTGCGTTGCTAGTGTCTGGTTGTGGTTGTAATAAGAACATGAAGAACATGTTGCTGTGGATCACATGACTTTTTTTTTTGAACCGGAAGATCACATGACTTTCATGGCTTTAAGTTCCTGAACTCTGAAATCTGGACCCCTTTTTAAGCTCTGAACTCATCATTCTTGCATTTACATCTGGTGTTGATCTTATTGATGTGATGCAGTCCTGCTGAAATAGTCAATGTAGATTCATGACTGACTGATTGCGTTTATGGTGTGTATGTTGTTAACAAGCTGAAGGTCGTGTGGTGTCTTTCCAGTTAGACGAAGTGTGCTTTATTGTAGCGTGTAGTGCTGCTGGATGATTGATGAACTGAAACATTCTGCATTTAGCAACTAGCGAGCCAAAGGTGATGACTGAGTTTCTGTAGACCTGTTTTTTTATGCCCATGGTCGTTCTTCAATTGCACTTGATTTTCACATTAGCTGGATCATAATCTGAGCAGACTACTCAAAAGTACAAAGTTCATCTTCGCTATGACGCTTTGCCACTAGGATTTTCTTTGTATGATTTGTTTACAAATCCTGTAATCTAGTCAAAAGAAAAGCCAAAATTTTTCTTTGTATGATTTGTTTACAAATCCTCTAATCTAGTCAAAGAAAAGCCAAATTTATCCCTCCTGGTCCCCTACATCACGTAGCTATGTGGCCCGCAAGCAGATGAAAGCAGCCCCGTCAGCCGACGCCGACGCCGACGCCAACACATCCTGCTCCTCCCTCGCCGGCGCCGGCGCCGGCGAGGCCGCACCGCCGCTGCCCCGTGGCCGCAGGCACACGGTGCCGCACTGCCGCCGCCCCGTGGCCGCAGGCACACGGTGCCGCACTGCCGCCGCCTCCCCTTCCGGCATTGCCGGACGGCTGGGCTACTGTCCCCGCCGCCTTCCCAAT

In an embodiment, a nucleic acid construct is provided comprising aubiquitin promoter as described herein and a 3′-UTR, wherein the 3′-UTRis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identical to SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, or SEQ ID NO:36. In an embodiment, a nucleic acid construct isprovided comprising a ubiquitin promoter as described herein and the3′-UTR wherein the ubiquitin promoter and 3′-UTR are both operablylinked to opposite ends of a polylinker. In an embodiment, a geneexpression cassette is provided comprising a ubiquitin promoter asdescribed herein and a 3′-UTR, wherein the ubiquitin promoter and 3′-UTRare both operably linked to opposite ends of a non-ubiquitin transgene.In one embodiment the a 3′-UTR, consists of SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, or SEQ ID NO:36. In one embodiment, a gene expressioncassette is provided comprising a ubiquitin promoter as described hereinand a 3′-UTR, wherein the ubiquitin promoter comprises SEQ ID NO: 2A andthe 3′-UTR comprises SEQ ID NO: 4 wherein the promoter and 3′-UTR areoperably linked to opposite ends of a non-ubiquitin transgene. In oneembodiment the a 3′-UTR, consists of SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, or SEQ ID NO:36. In one embodiment the promoter consists of SEQ IDNO: 2A, 15 or 40 and the 3′-UTR, consists of SEQ ID NO:4. In anillustrative embodiment, a gene expression cassette comprises aubiquitin 3′-UTR that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water usefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof. In afurther embodiment the transgene is operably linked to a ubiquitinpromoter and a 3′-UTR from the same ubiquitin gene isolated from Panicumvirgatum, Brachypodium distachyon, or Setaria italica.

In one embodiment a vector is provided comprising a first transgeneand/or polylinker and a second transgene and/or polylinker wherein thefirst transgene and/or polylinker is operably linked to a promotercomprising a sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:35 and operably linked to a3′-UTR, comprising a sequence selected from the group consisting of SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36 and the secondtransgene and/or polylinker is operably linked to a promoter comprisinga sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, and SEQ ID NO:35 and operably linked to a 3′-UTR,comprising a sequence selected from the group consisting of SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36, further wherein the promoterof the first transgene and/or polylinker and second transgene and/orpolylinker are derived from Ubi genes from different plant species. In afurther embodiment the vector is provided with a third transgene and/orpolylinker wherein the third transgene and/or polylinker polylinker isoperably linked to a promoter comprising a sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ IDNO:35 and operably linked to a 3′-UTR, comprising a sequence selectedfrom the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, orSEQ ID NO:36, further wherein the promoter of the third transgene and/orpolylinker is derived from Ubi genes from a different plant species fromthe promoter of the first and second transgene and/or polylinker.

Transgene expression may also be regulated by an intron region locateddownstream of the promoter sequence. Both a promoter and an intron canregulate transgene expression. While a promoter is necessary to drivetranscription, the presence of an intron can increase expression levelsresulting in mRNA transcript for translation and protein synthesis. Anintron gene region aids stable expression of a transgene.

In an embodiment, a nucleic acid construct is provided comprising aubiquitin promoter as described herein and an intron. In one embodimentthe intron is operably linked to the 3′ end of the promoter. In anembodiment, a nucleic acid construct is provided comprising a ubiquitinintron operably linked to the 3′ end of a ubiquitin promoter isolatedfrom Panicum virgatum, Brachypodium distachyon or Setaria italica or aderivative of such promoter sequence. In an embodiment, the ubiquitinintron is a Panicum virgatum, Brachypodium distachyon or Setaria italicaubiquitin intron, or a derivative of such intron sequence.

In an embodiment, an intron can be the Brachypodium distachyonubiquitin1 C intron.

(SEQ ID NO: 7) GTATGCAGCCTCGCTTCCTCCTCGCTACCGTTTCAATTCTGGAGTAGGTCGTAGAGGATACCATGTTGATTTGACAGAGGGAGTAGATTAGATACTTGTAGATCGAAGTGCGGATGTTCCATGGTAGATGATACCATGTTGATTTCGATTAGATCGGATTAAATCTTTGTAGATCGAAGTGCGCATGTTCCATGAATTGCCTGTTACCAGTAGATTCAAGTTTTTCTGTGTTATAGAGGTGGGATCTACTCGTTGAGATGATTAGCTCCTAGAGGACACCATGCCGTTTTGGAAAATAGATCAGAACCGTGTAGATCGATGTGAGCATGTGTTCCTGTAGATCCAAGTTCTTTCGCATGTTACTAGTTGTGATCTATTGTTTGTGTAATACGCTCTCGATCTATCCGTGTAGATTTCACTCGATTACTGTTACTGTGGCTTGATCGTTCATAGTTGTTCGTTAGGTTTGATCGAACAGTGTCTGAACCTAATTGGATATGTATTCTTGATCTATCAACGTGTAGGTTTCAGTCATGTATTTATGTACTCCCTCCGTCCCAAATTAACTGACGTGGATTTTGTATAAGAATCTATACAAATCCATGTCAGTTAATTCGGGATGGAGTACCATATTCAATAATTTGTTTATTGCTGTCCACTTATGTACCATATGTTTGTTGTTCCTCATGTGGATTCTACTAATTATCATTGATTGGTGATCTTCTATTTTGCTAGTTTCCTAGCTCAATCTGGTTATTCATGTAGATGTGTTGTTGAAATCGGAGACCATGCTTGTTATTAGATAGTTTATTGCTTATCAGTTTCATGTTCTGGTTGATGCAACACATATTCATGTTCGCTATCTGGTTGCTGCTTGATATTCTCTGATTTACATTCATTATAAGAATATATTCTGCTCTGGTTGTTGCTTCTCATGACTTTACCTACTCGGTAGGTGACTTACCTTTTGGTTTACAATT GTCAACTATGCAGIn an embodiment, an intron can be the Brachypodium distachyon ubiquitin1 (Ubi1) intron

(SEQ ID NO: 8) GTATGTAGCCTCTCGATTCCTCCTCAGCCCTGCCCTCGATTTGGTGTACGCGTTGAGATGATGATCTCGTAGATGTCTAGATGACACCATGTCGATTTGAAATAGATCAGATCCGTGTAGATCGATGAGCTCCTGTGTACCTGTGGATTCAAGTTATTTTCGCATGCTATTGTTGTGATCTACTAGATCTAGTGTGTGTATTCTATGCTATCGATTTCTCCGTGTAGATTTCACTCGATTACTGTTACTGTGGCTTGATCGGCCATAGATGTTGGTTAAGGTTTGATCGGTTAGTGTTTGAACCTGCGTGGATATCTAGCATCCATCTATTATCGTGTAGGTTTCGAACAAACAAGCACTATTATTGTACTGATGGTTCGTCTATGGTTGGTTTTGACCGTTTTAGTGTTGAACGAGCCTTCTGTATTTGTTTATTGCTGTCCAGTGATGTACCATGTTCGTTGAGTGTCGGATTATACTAATTATTGTTGATTGATAATCTTGTAGTTTGCTTTTCCTAATTTATTTATCGTAGTCCTGATTTGCCTCAGCTGTGCCTCACCCGTGCGATGGTCAATCAACTTGTTAGCCCAATCTGCTTAATCATGTACATTTGTTGTTAGAATCAGAGATCAAGCCAATTAGCTATCTTATTGCTTATCTGTTCCATGTTCTGATCGATGTAACAGTCTACACTTTTGCTCTGTGCTACTTGATTAAAACATTCTGACTTAAATTCATGATTGGAAGTTTCAGATCTGATTGTTGCCTTACTTGACTAATATCTATTCATGTGACACCTCTCTGTCTTGGTAACTTACCGCTGTTTGTTT GTAATTTCTGACTATGCAGIn an embodiment, an intron can be the Setaria italica ubiquitin 2(Ubi2) intron 1

(SEQ ID NO: 9) GTCACGGGTTCCTTCCCCACCTCTCCTCTTCCCCACCGCCATAAATAGIn an embodiment, an intron can be the Setaria italica ubiquitin 2(Ubi2) intron 2

(SEQ ID NO: 10) GTACGGCGATCGTCTTCCTCCTCTAGATCGGCGTGATCTGCAAGTAGTTGATTTGGTAGATGGTTAGGATCTGTGCACTGAAGAAATCATGTTAGATCCGCGATGTTTCTGTTCGTAGATGGCTGGGAGGTGGAATTTTTGTGTAGATCTGATATGTTCTCCTGTTTATCTTGTCACGCTCCTGCGATTTGTGGGGATTTTAGGTCGTTGATCTGGGAATCGTGGGGTTGCTTCTAGGCTGTTCGTAGATGAGGTCGTTCTCACGGTTTACTGGATCATTGCCTAGTAGATCAGCTCGGGCTTTCGTCTTTGTATATGGTGCCCATACTTGCATCTATGATCTGGTTCCGTGGTGTTACCTAGGTTTCTGCGCCTGATTCGTCCGATCGATTTTGTTAGCATGTGGTAAACGTTTGGTCATGGTCTGATTTAGATTAGAGTCGAATAGGATGATCTCGATCTAGCTCTTGGGATTAATATGCATGTGTCACCAATCTGTTCCGTGGTTAAGATGATGAATCTATGCTTAGTTAATGGGTGTAGATATATATGCTGCTGTTCCTCAATGATGCCGTAGCTTTTACCTGAGCAGCATGGATCCTCCTGTTACTTAGGTAGATGCACATGCTTATAGATCAAGATATGTACTGCTACTGTTGGAATTCTTTAGTATACCTGATGATCATCCATGCTCTTGTTACTTGTTTTGGTATACTTGGATGATGGCATGCTGCTGCTTTTTGTTGATTTGAGCCCATCCATATCTGCATATGTCACATGATTAAGATGATTACGCTGTTTCTGTATGATGCCATAGCTTTTATGTGAGCAACATGCATCCTCCTGGTTATATGCATTAATAGATGGAAGATATCTATTGCTACAATTTGATGATTATTTTGTACATACGATGATCAAGCATGCTCTTCATACTTTGTTGATATACTTGGATAATGAAATGCTGCTGCACGTTCATTCTATAGCACTAATGATGTGATGAACACGCACGACCTGTTTGTGGCATCTGTTTGAATGTGTTGTTGCTGTTCACTAGAGACTGTTTTATTAACCTACTGCTAGATACTTACCCTTCTGTCTGT TTATTCTTTTGCAGIn an embodiment, an intron can be the Panicum virgatum (Switchgrass)ubiquitin intron.

(SEQ ID NO: 37) GTACTCCTACCTAATCCTCCTTAACTGATCTCTCCTCTATCACGTTGGTAATCTTCGAATGATCTGCTGCCTGGCTCGCTGTTCCCCCTCGTTATGCACTGTTTCCATCACGAGTTTTTTTTTTCATCATCTAATCTATGCGGTTGCGGAAGAATTGTGGCTAGTGGAGTAGTTTTCTGTGCTTGATCGGTAGATTCGATGTGTGGGTGTATGGATGTTTTCTGAAAAGTTGCTGGATTAGTTTACGCTTTCAGGCCGCAGGTCTGTTCGAAATTGATTATGAAGTCTATATGCTTTGGATCTATCGATTTCCAGTTTTATTCAGATGTAGGCCAAAAAATTGTCGGCATTTGTGTGGAATTAGTTGGCCTTTAGGTCTGCACATTCATGGTGACGGCACAGTTGCTGCTGGCTGTTGCGTGGGACGAGTTATTATAGTTGTTTTTGTTTTTCCCTGATTGATTCACATTTTCAATGATAACTAGCCTTTGTCACCTAACCAAGTCCAGGTTGATCCTATCTGTGTTCTTCAGCTACCAGTTTGCATAGATGATGGTGTATTCGATTGCTTTAGTAGGCCTTCTGATTTCACATCTAATTCTGTCATGAATATAGATAACTTTACATGCTTTTGATATACTTTATATTTGAACTGTTCACTGTCCAGCCTATTTTGGATAATTGAGTGCATTGGCTTTTGATGCCTGAATTATTCACATGTTCCTGGATAATTGACCTGTGTCACCTAGTTGACTGTTTTTTGAGGTGCCACCCGTCTGTTCAGCTGATTTGTGTATTCGATTGCTCTAGTTAATCTTTTGATTATGCAGCTAGTGCTTTGTCATATGTAGCTTTATAGGCTTCTGATGTCCTTGGATATAGTTCAGTCTACTTGTCAAGTTGCTTTACAAGTAGTAGCTCTGATTCTATTTGGCTTCCTGAGTCAGAGCTTTGCAAATTGCTTGTTGTTACATTACATCATATTACTTGAATTGCAGTTATTTAATGGTTGGATTGTTGCTGTTTACTTCTACATTTTTTGCTGTTTTATATTATACTAAAATGTTTGTGTTGCTGCTTTTCAG

In an embodiment, a nucleic acid construct is provided comprising aubiquitin promoter as described herein and an intron, wherein the intronis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identical to SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, or SEQ ID NO:37. In an embodiment, a nucleic acidconstruct is provided comprising a ubiquitin promoter as describedherein, an intron sequence and a polylinker wherein the promoter andintron are operably linked to a polylinker. In an embodiment, a geneexpression cassette is provided comprising a ubiquitin promoter asdescribed herein, an intron sequence and a non-ubiquitin transgenewherein the promoter and intron are operably linked to the 5′ end of thetransgene. Optionally the construct further comprises a 3′-UTR that isoperably linked to the 3′ end of the non-ubiquitin transgene orpolylinker. In one embodiment the promoter and 3′-UTR sequences areselected from those described herein and the intron sequence consists ofSEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37. Inan embodiment, a gene expression cassette comprises a ubiquitin intronthat is operably linked to a promoter, wherein the promoter is a Panicumvirgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter,or a promoter that originates from a plant (e.g., Zea mays ubiquitin 1promoter), a virus (e.g., Cassava vein mosaic virus promoter) or abacteria (e.g., Agrobacterium tumefaciens delta mas). In an illustrativeembodiment, a gene expression cassette comprises a ubiquitin intron thatis operably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water us efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof.

Transgene expression may also be regulated by a 5′-UTR region locateddownstream of the promoter sequence. Both a promoter and a 5′-UTR canregulate transgene expression. While a promoter is necessary to drivetranscription, the presence of a 5′-UTR can increase expression levelsresulting in mRNA transcript for translation and protein synthesis. A5′-UTR gene region aids stable expression of a transgene.

In an embodiment, a nucleic acid construct is provided comprising aubiquitin promoter as described herein and a 5′-UTR. In one embodimentthe 5′-UTR is operably linked to the 3′ end of the promoter. In anembodiment, a nucleic acid construct is provided comprising a ubiquitina 5′-UTR operably linked to the 3′ end of a ubiquitin promoter isolatedfrom Panicum virgatum, Brachypodium distachyon or Setaria italica or aderivative of such promoter sequence. In a further embodiment the 3′ endof the 5′-UTR is operably linked to the 5′ end of a ubiquitin intronfrom Panicum virgatum, Brachypodium distachyon or Setaria italica, asdescribed herein.

In an embodiment, a 5′-UTR can be the Brachypodium distachyon ubiquitin1C (Ubi1C) 5′-UTR.

(SEQ ID NO: 11) CCGATCGAAAAGTCCCCGCAAGAGCAAGCGACCGATCTCGTGAATCTCCG TCAAG

In an embodiment, a 5′-UTR can be the Brachypodium distachyon ubiquitin1 (Ubi1) 5′-UTR.

(SEQ ID NO: 12) CCAATCCAGCACCCCCGATCCCGATCGAAAATTCTCCGCAACAGCAAGCGATCGATCTAGCGAATCCCCGTCAAG

In an embodiment, a 5′-UTR can be the Setaria italica ubiquitin 2 (Ubi2)5′-UTR1

(SEQ ID NO: 13) AGAAATATCAACTGGTGGGCCACGCACATCAGCGTCGTGTAACGTGGACGGAGGAGCCCCGTGACGGCGTCGACATCGAACGGCCACCAACCACGGAACCACCCGTCCCCACCTCTCGGAAGCTCCGCTCCACGGCGTCGACATCTAACGGCTACCAGCAGGCGTACGGGTTGGAGTGGACTCCTTGCCTCTTTGCGCTGGCGGCTTCCGGAAATTGCGTGGCGGAGACGAGGCGGGCTCGTCTCACACG GCACGGAAGAC

In an embodiment, a 5′-UTR can be the Setaria italica ubiquitin 2 (Ubi2)5′-UTR2

(SEQ ID NO: 14) CCGACCCCCTCGCCTTTCTCCCCAATCTCATCTCGTCTCGTGTTGTTCGGAGCACACCACCCGCCCCAAATCGTTCTTCCCGCAAGCCTCGGCGATCCTT CACCCGCTTCAAG

In an embodiment, a 5′-UTR can be the Panicum virgatum (Switchgrass)ubiquitin 5′-UTR.

(SEQ ID NO: 38) CAAGTTCGCGATCTCTCGATTTCACAAATCGCCGAGAAGACCCGAGCAGAGAAGTTCCCTCCGATCGCCTTGCCAAG.

In an embodiment, a nucleic acid construct is provided comprising aubiquitin promoter as disclosed herein and a 5′-UTR, wherein the 5′-UTRis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identical to SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, or SEQ ID NO:38. In an embodiment, a nucleic acidconstruct is provided comprising ubiquitin promoter, wherein thepromoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, or SEQ ID NO:35, and a 5′-UTR operably linked to apolylinker. In an embodiment, a gene expression cassette is providedcomprising a ubiquitin promoter, wherein the promoter is at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ IDNO:35, and a 5′-UTR sequences operably linked to a non-ubiquitintransgene. Optionally, the construct can further comprise a ubiquitinintron as disclosed herein operably linked to the 3′ end of the 5′-UTRand the 5′ end of the non-ubiquitin transgene and optionally furthercomprising a 3′-UTR that is operably linked to the 3′ end of thenon-ubiquitin transgene. In one embodiment the promoter, intron and3′-UTR sequences are selected from those described herein and the 5′-UTRsequence consists of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, or SEQ ID NO:38. In one embodiment the 3′-UTR consists of SEQ IDNO:11.

In an embodiment, a gene expression cassette comprises a ubiquitin5′-UTR that is operably linked to a promoter, wherein the promoter is aPanicum virgatum, Brachypodium distachyon or Setaria italica ubiquitinpromoter, or a promoter that originates from a plant (e.g., Zea maysubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic viruspromoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). Inan illustrative embodiment, a gene expression cassette comprises aubiquitin 5′-UTR that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water usefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof.

In one embodiment a nucleic acid construct is provided comprising apromoter and a polylinker and optionally one or more of the followingelements:

a) a 5′ untranslated region;

b) an intron; and

c) a 3′ untranslated region,

wherein

the promoter consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQID NO:35 or a sequence having 98% sequence identity with SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35;

the 5′ untranslated region consists of SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, or SEQ ID NO:38 or a sequence having 98%sequence identity with SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, or SEQ ID NO:38

the intron consists of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, or SEQ ID NO:37 or a sequence having 98% sequence identity withSEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37

the 3′ untranslated region consists of SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, or SEQ ID NO:36 or a sequence having 98% sequence identity withSEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36; further whereinsaid promoter is operably linked to said polylinker and each optionalelement, when present, is also operably linked to both the promoter andthe polylinker.

In one embodiment a nucleic acid construct is provided comprising apromoter and a non-ubiquitin transgene and optionally one or more of thefollowing elements:

a) a 5′ untranslated region;

b) an intron; and

c) a 3′ untranslated region,

wherein

the promoter consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQID NO:35 or a sequence having 98% sequence identity with SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35;

the 5′ untranslated region consists of SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, or SEQ ID NO:38 or a sequence having 98%sequence identity with SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, or SEQ ID NO:38

the intron consists of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, or SEQ ID NO:37 or a sequence having 98% sequence identity withSEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37

the 3′ untranslated region consists of SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, or SEQ ID NO:36 or a sequence having 98% sequence identity withSEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36; further whereinsaid promoter is operably linked to said transgene and each optionalelement, when present, is also operably linked to both the promoter andthe transgene. In a further embodiment a transgenic cell is providedcomprising the nucleic acid construct disclosed immediately above. Inone embodiment the transgenic cell is a plant cell, and in a furtherembodiment a plant is provided wherein the plant comprises saidtransgenic cells.

In accordance with one embodiment transgene expression is regulated by apromoter operably linked to an intron and 5′-UTR region, wherein theintron and 5′-UTR region are located downstream of the promotersequence. A promoter operably linked to an intron and 5′-UTR region canbe used to drive transgene expression. While a promoter is necessary todrive transcription, the presence of the intron and 5′-UTR can increaseexpression levels resulting in mRNA transcript for translation andprotein synthesis.

In an embodiment, a gene expression cassette comprises a promoteroperably linked to a 5′-UTR and intron region. In an embodiment, a geneexpression cassette comprises a ubiquitin promoter operably linked to aubiquitin 5′-UTR and ubiquitin intron. In an embodiment, the ubiquitinpromoter operably linked to a 5′-UTR and intron region is a Panicumvirgatum, Brachypodium distachyon or Setaria italica ubiquitin promoteroperably linked to an intron and 5′-UTR.

In an embodiment, a promoter operably linked to a 5′-UTR and intron canbe the Brachypodium distachyon ubiquitin1 C (Ubi1C) promoter operablylinked to an intron and 5′-UTR. In one embodiment the promoter comprisesor consists of the sequence of SEQ ID NO: 15:

(SEQ ID NO: 15) CTGCTCGTTCAGCCCACAGTAACACGCCGTGCGACATGCAGATGCCCTCCACCACGCCGACCAACCCCAAGTCCGCCGCGCTCGTCCACGGCGCCATCCGCATCCGCGCGTCAACGTCATCCGGAGGAGGCGAGCGCGATGTCGACGGCCACGGCGGCGGCGGACACGACGGCGACGCCCCGACTCCGCGCGCGCGTCAAGGCTGCAGTGGCGTCGTGGTGGCCGTCCGCCTGCACGAGATCCCCGCGTGGACGAGCGCCGCCTCCACCCAGCCCCTATATCGAGAAATCAACGGTGGGCTCGAGCTCCTCAGCAACCTCCCCACCCCCCCTTCCGACCACGCTCCCTTCCCCCGTGCCCCTCTTCTCCGTAAACCCGAGCCGCCGAGAACAACACCAACGAAAGGGCGAAGAGAATCGCCATAGAGAGGAGATGGGCGGAGGCGGATAGTTTCAGCCATTCACGGAGAAATGGGGAGGAGAGAACACGACATCATACGGACGCGACCCTCTAGCTGGCTGGCTGTCCTAAAGAATCGAACGGAATCGCTGCGCCAGGAGAAAACGAACGGTCCTGAAGCATGTGCGCCCGGTTCTTCCAAAACACTTATCTTTAAGATTGAAGTAGTATATATGACTGAAATTTTTACAAGGTTTTTCCCCATAAAACAGGTGAGCTTATCTCATCCTTTTGTTTAGGATGTACGTATTATATATGACTGAATATTTTTTATTTTCATTGAATGAAGATTTTCGACCCCCCAAAAATAAAAAACGGAGGGAGTACCTTTGTGCCGTGTATATGGACTAGAGCCATCGGGACGTTTCCGGAGACTGCGTGGTGGGGGCGATGGACGCACAACGACCGCATTTTCGGTTGCCGACTCGCCGTTCGCATCTGGTAGGCACGACTCGTCGGGTTCGGCTCTTGCGTGAGCCGTGACGTAACAGACCCGTTCTCTTCCCCCGTCTGGCCATCCATAAATCCCCCCTCCATCGGCTTCCCTTTCCTCAATCCAGCACCCTGATTCCGATCGAAAAGTCCCCGCAAGAGCAAGCGACCGATCTCGTGAATCTCCGTCAAGGTATGCAGCCTCGCTTCCTCCTCGCTACCGTTTCAATTCTGGAGTAGGTCGTAGAGGATACCATGTTGATTTGACAGAGGGAGTAGATTAGATACTTGTAGATCGAAGTGCGGATGTTCCATGGTAGATGATACCATGTTGATTTCGATTAGATCGGATTAAATCTTTGTAGATCGAAGTGCGCATGTTCCATGAATTGCCTGTTACCAGTAGATTCAAGTTTTTCTGTGTTATAGAGGTGGGATCTACTCGTTGAGATGATTAGCTCCTAGAGGACACCATGCCGTTTTGGAAAATAGATCAGAACCGTGTAGATCGATGTGAGCATGTGTTCCTGTAGATCCAAGTTCTTTCGCATGTTACTAGTTGTGATCTATTGTTTGTGTAATACGCTCTCGATCTATCCGTGTAGATTTCACTCGATTACTGTTACTGTGGCTTGATCGTTCATAGTTGTTCGTTAGGTTTGATCGAACAGTGTCTGAACCTAATTGGATATGTATTCTTGATCTATCAACGTGTAGGTTTCAGTCATGTATTTATGTACTCCCTCCGTCCCAAATTAACTGACGTGGATTTTGTATAAGAATCTATACAAATCCATGTCAGTTAATTCGGGATGGAGTACCATATTCAATAATTTGTTTATTGCTGTCCACTTATGTACCATATGTTTGTTGTTCCTCATGTGGATTCTACTAATTATCATTGATTGGTGATCTTCTATTTTGCTAGTTTCCTAGCTCAATCTGGTTATTCATGTAGATGTGTTGTTGAAATCGGAGACCATGCTTGTTATTAGATAGTTTATTGCTTATCAGTTTCATGTTCTGGTTGATGCAACACATATTCATGTTCGCTATCTGGTTGCTGCTTGATATTCTCTGATTTACATTCATTATAAGAATATATTCTGCTCTGGTTGTTGCTTCTCATGACTTTACCTACTCGGTAGGTGACTTACCTTTTGGTTTACAATTGTCAACTATGCAG

In an embodiment, a promoter operably linked to 5′-UTR and intron can bethe Brachypodium distachyon ubiquitin 1 (Ubi1) promoter operably linkedto a 5′-UTR and intron. In one embodiment the promoter comprises orconsists of the sequence of SEQ ID NO: 16:

(SEQ ID NO: 16) GGCGTCAGGACTGGCGAAGTCTGGACTCTGCAGGGCCGAACTGCTGAAGACGAAGCAGAGGAAGAGAAAGGGAAGTGTTCGACTTGTAATTTGTAGGGGTTTTTTTTAGAGGAACTTGTAATTTGTAGGTGGGCTGGCCTCGTTGGAAAAACGATGCTGGCTGGTTGGGCTGGGCCGATGTACGCTTGCAAACAACTTGTGGCGGCCCGTTCTGGACGAGCAGGAGTTTCTTTTTTGTTCTCACTTTTCTGGTCTTCTTTAGTTACGGAGTACCTTTTGTTTTTTAAAGGAGTTACCTTTTTTTTAGGAATTCTTTAGTTACCTTTCGCTTGCTCTCAAAAAATATTTAACTTTCGCTTTTTTTCATTTTAATTTTTGCAACTATTTACGAGTTTCATGAATGCTTATTTTCCAGCATATCATTATTTGCAAGTATTTTTATGCCGTATGTATTGGACGAGAGCCATCGGGACTGTTCCAGAGACTGCGTGGTGGGGACGGCTCCCAACCGCCTTTTCTATCTCTGTTCGCATCCGGTGGCCGACTTGGCTCGCGCGTGAGCCGTGACGTAACAGACTTGGTCTCTTCCCCATCTGGCCATCTATAAATTCCCCCATCGATCGACCCTCCCTTTCCCCAATCCAGCACCCCCGATCCCGATCGAAAATTCTCCGCAACAGCAAGCGATCGATCTAGCGAATCCCCGTCAAGGTATGTAGCCTCTCGATTCCTCCTCAGCCCTGCCCTCGATTTGGTGTACGCGTTGAGATGATGATCTCGTAGATGTCTAGATGACACCATGTCGATTTGAAATAGATCAGATCCGTGTAGATCGATGAGCTCCTGTGTACCTGTGGATTCAAGTTATTTTCGCATGCTATTGTTGTGATCTACTAGATCTAGTGTGTGTATTCTATGCTATCGATTTCTCCGTGTAGATTTCACTCGATTACTGTTACTGTGGCTTGATCGGCCATAGATGTTGGTTAAGGTTTGATCGGTTAGTGTTTGAACCTGCGTGGATATCTAGCATCCATCTATTATCGTGTAGGTTTCGAACAAACAAGCACTATTATTGTACTGATGGTTCGTCTATGGTTGGTTTTGACCGTTTTAGTGTTGAACGAGCCTTCTGTATTTGTTTATTGCTGTCCAGTGATGTACCATGTTCGTTGAGTGTCGGATTATACTAATTATTGTTGATTGATAATCTTGTAGTTTGCTTTTCCTAATTTATTTATCGTAGTCCTGATTTGCCTCAGCTGTGCCTCACCCGTGCGATGGTCAATCAACTTGTTAGCCCAATCTGCTTAATCATGTACATTTGTTGTTAGAATCAGAGATCAAGCCAATTAGCTATCTTATTGCTTATCTGTTCCATGTTCTGATCGATGTAACAGTCTACACTTTTGCTCTGTGCTACTTGATTAAAACATTCTGACTTAAATTCATGATTGGAAGTTTCAGATCTGATTGTTGCCTTACTTGACTAATATCTATTCATGTGACACCTCTCTGTCTTGGTAACTTACCGCTGTTTGTTTGTAATT TCTGACTATGCAG

In an embodiment, a promoter operably linked to a 5′-UTR and intron canbe the Setaria italica ubiquitin 2 (Ubi2) promoter operably linked to a5′-UTR and intron. In one embodiment the promoter comprises or consistsof the sequence of SEQ ID NO: 17:

(SEQ ID NO: 17) TGCGTCTGGACGCACAAGTCATAGCATTATCGGCTAAAATTTCTTAATTTCTAAATTAGTCATATCGGCTAAGAAAGTGGGGAGCACTATCATTTCGTAGAACAAGAACAAGGTATCATATATATATATATATATAATATTTAAACTTTGTTAAGTGGAATCAAAGTGCTAGTATTAATGGAGTTTCATGTGCATTAAATTTTATGTCACATCAGCAATTTTGTTGACTTGGCAAGGTCATTTAGGGTGTGTTTGGAAGACAGGGGCTATTAGGAGTATTAAACATAGTCTAATTACAAAACTAATTGCACAACCGCTAAGCTGAATCGCGAGATGGATCTATTAAGCTTAATTAGTCCATGATTTGACAATGTGGTGCTACAATAACCATTTGCTAATGATGGATTACTTAGGTTTAATAGATTCGTCTCGTGATTTAGCCTATGGGTTCTGCTATTAATTTTGTAATTAGCTCATATTTAGTTCTTATAATTAGTATCCGAACATCCAATGTGACATGCTAAAGTTTAACCCTGGTATCCAAATGAAGTCTTATGAGAGTTTCATCACTCCGGTGGTATATGTACTTAGGCTCCGTTTTCTTCCACCGACTTATTTTTAGCACCCGTCACATTGAATGTTTAGATACTAATTAGAAGTATTAAACGTAGACTATTTACAAAATCCATTACATAAGACGAATCTAAACGGCGAGACGAATCTATTAAACCTAATTAGTCCATGATTTGACAATGTGTTGCTACAGTAAACATTTGCTAATGATGGATTAATTAGGCTTAATAGATTCGTCTCGCCGTTTAGCCTCCACTTATGTAATGGGTTTTCTAAACAATCTACGTTTAATACTCCTAATTAGTATCTAAATATTCAATGTGACACGTGCTAAAAATAAGTCAGTGGAAGGAAGAGAACGTCCCCTTAGTTTTCCATCTTATTAATTGTACGATGAAACTGTGCAGCCAGATGATTGACAATCGCAATACTTCAACTAGTGGGCCATGCACATCAGCGACGTGTAACGTCGTGAGTTGCTGTTCCCGTAGAGAAATATCAACTGGTGGGCCACGCACATCAGCGTCGTGTAACGTGGACGGAGGAGCCCCGTGACGGCGTCGACATCGAACGGCCACCAACCACGGAACCACCCGTCCCCACCTCTCGGAAGCTCCGCTCCACGGCGTCGACATCTAACGGCTACCAGCAGGCGTACGGGTTGGAGTGGACTCCTTGCCTCTTTGCGCTGGCGGCTTCCGGAAATTGCGTGGCGGAGACGAGGCGGGCTCGTCTCACACGGCACGGAAGACGTCACGGGTTCCTTCCCCACCTCTCCTCTTCCCCACCGCCATAAATAGCCGACCCCCTCGCCTTTCTCCCCAATCTCATCTCGTCTCGTGTTGTTCGGAGCACACCACCCGCCCCAAATCGTTCTTCCCGCAAGCCTCGGCGATCCTTCACCCGCTTCAAGGTACGGCGATCGTCTTCCTCCTCTAGATCGGCGTGATCTGCAAGTAGTTGATTTGGTAGATGGTTAGGATCTGTGCACTGAAGAAATCATGTTAGATCCGCGATGTTTCTGTTCGTAGATGGCTGGGAGGTGGAATTTTTGTGTAGATCTGATATGTTCTCCTGTTTATCTTGTCACGCTCCTGCGATTTGTGGGGATTTTAGGTCGTTGATCTGGGAATCGTGGGGTTGCTTCTAGGCTGTTCGTAGATGAGGTCGTTCTCACGGTTTACTGGATCATTGCCTAGTAGATCAGCTCGGGCTTTCGTCTTTGTATATGGTGCCCATACTTGCATCTATGATCTGGTTCCGTGGTGTTACCTAGGTTTCTGCGCCTGATTCGTCCGATCGATTTTGTTAGCATGTGGTAAACGTTTGGTCATGGTCTGATTTAGATTAGAGTCGAATAGGATGATCTCGATCTAGCTCTTGGGATTAATATGCATGTGTCACCAATCTGTTCCGTGGTTAAGATGATGAATCTATGCTTAGTTAATGGGTGTAGATATATATGCTGCTGTTCCTCAATGATGCCGTAGCTTTTACCTGAGCAGCATGGATCCTCCTGTTACTTAGGTAGATGCACATGCTTATAGATCAAGATATGTACTGCTACTGTTGGAATTCTTTAGTATACCTGATGATCATCCATGCTCTTGTTACTTGTTTTGGTATACTTGGATGATGGCATGCTGCTGCTTTTTGTTGATTTGAGCCCATCCATATCTGCATATGTCACATGATTAAGATGATTACGCTGTTTCTGTATGATGCCATAGCTTTTATGTGAGCAACATGCATCCTCCTGGTTATATGCATTAATAGATGGAAGATATCTATTGCTACAATTTGATGATTATTTTGTACATACGATGATCAAGCATGCTCTTCATACTTTGTTGATATACTTGGATAATGAAATGCTGCTGCACGTTCATTCTATAGCACTAATGATGTGATGAACACGCACGACCTGTTTGTGGCATCTGTTTGAATGTGTTGTTGCTGTTCACTAGAGACTGTTTTATTAACCTACTGCTAGATACTTACCCTTCTGTCTGTTTATTCTTTTGCAG

In an embodiment, a promoter operably linked to a 5′-UTR and intron canbe the Panicum virgatum (Switchgrass) ubiquitin promoter operably linkedto a 5′-UTR and intron. In one embodiment the promoter comprises orconsists of the sequence of SEQ ID NO: 39:

(SEQ ID NO: 39) TTGAATTTTAATTTCAAATTTTGCAGGGTAGTAGTGGACATCACAATACATATTTAGAAAAAGTTTTATAATTTTCCTCCGTTAGTTTTCATATAATTTTGAACTCCAACGATTAATCTATTATTAAATATCCCGATCTATCAAAATAATGATAAAAATTTATGATTAATTTTTCTAACATGTGTTATGGTGTGTACTATCGTCTTATAAAATTTCAACTTAAAACTCCACCTATACATGGAGAAATGAAAAAGACGAATTACAGTAGGGAGTAATTTGAACCAAATGGAATAGTTTGAGGGTAAAATGAACTAAACAATAGTTTAGGAGGTTATTCAGATTTTAGTTATAGTTGAGAGGAGTAATTTAGACTTTTTCCTATCTTGAATTGTTGACGGCTCTCCTATCGGATATCGGATGGAGTCTTTCAGCCCAACATAACTTCATTCGGGCCCAAACGTTCGTCCATCCAGCCTAGGGAGAACATTTTGCCCATGATATCTGTTTTTCTTTTTTTCTATTTTCACTGGTATTATAGGAGGGAAATATACAACGTGTTCACCTTTGGTTTCATTCTTGTTCCATCTGAATTTATCTAAAACTGTGTTTGAACTTCGTAAGAATTTTGTTCGATCTGTCCGGTACATCGTGTTGATAGGTGGCCTCCGAGATTCTTCTTTTTAACCGGCAAAGTAAAATAATCTCAGCTCCAGCCTAACGTCAATTATCAGAGAGAGAAAAAAATATTTTTTTATGATTGATCGGAAACCAACCGCCTTACGTGTCGATCCTGGTTCCTGGCCGGCACGGCGGAGGAAAGCGACCGACCTCGCAACGCCGGCGCACGGCGCCGCCGTGTTGGACTTGGTCTCCCGCGACTCCGTGGGCCTCGGCTTATCGCCGCCGCTCCATCTCAACCGTCCGCTTGGACACGTGGAAGTTGATCCGTCGCGCACCAGCCTCGGAGGTAACCTAACTGCCCGTACTATAAATCCGGGATCCGGCCTCTCCAATCCCCATCGCCACAAGTTCGCGATCTCTCGATTTCACAAATCGCCGAGAAGACCCGAGCAGAGAAGTTCCCTCCGATCGCCTTGCCAAGGTACTCCTACCTAATCCTCCTTAACTGATCTCTCCTCTATCACGTTGGTAATCTTCGAATGATCTGCTGCCTGGCTCGCTGTTCCCCCTCGTTATGCACTGTTTCCATCACGAGTTTTTTTTTTCATCATCTAATCTATGCGGTTGCGGAAGAATTGTGGCTAGTGGAGTAGTTTTCTGTGCTTGATCGGTAGATTCGATGTGTGGGTGTATGGATGTTTTCTGAAAAGTTGCTGGATTAGTTTACGCTTTCAGGCCGCAGGTCTGTTCGAAATTGATTATGAAGTCTATATGCTTTGGATCTATCGATTTCCAGTTTTATTCAGATGTAGGCCAAAAAATTGTCGGCATTTGTGTGGAATTAGTTGGCCTTTAGGTCTGCACATTCATGGTGACGGCACAGTTGCTGCTGGCTGTTGCGTGGGACGAGTTATTATAGTTGTTTTTGTTTTTCCCTGATTGATTCACATTTTCAATGATAACTAGCCTTTGTCACCTAACCAAGTCCAGGTTGATCCTATCTGTGTTCTTCAGCTACCAGTTTGCATAGATGATGGTGTATTCGATTGCTTTAGTAGGCCTTCTGATTTCACATCTAATTCTGTCATGAATATAGATAACTTTACATGCTTTTGATATACTTTATATTTGAACTGTTCACTGTCCAGCCTATTTTGGATAATTGAGTGCATTGGCTTTTGATGCCTGAATTATTCACATGTTCCTGGATAATTGACCTGTGTCACCTAGTTGACTGTTTTTTGAGGTGCCACCCGTCTGTTCAGCTGATTTGTGTATTCGATTGCTCTAGTTAATCTTTTGATTATGCAGCTAGTGCTTTGTCATATGTAGCTTTATAGGCTTCTGATGTCCTTGGATATAGTTCAGTCTACTTGTCAAGTTGCTTTACAAGTAGTAGCTCTGATTCTATTTGGCTTCCTGAGTCAGAGCTTTGCAAATTGCTTGTTGTTACATTACATCATATTACTTGAATTGCAGTTATTTAATGGTTGGATTGTTGCTGTTTACTTCTACATTTTTTGCTGTTTTATATTATACTAAAATGTTTGTGTTGCTGCTTTTCAG

In an embodiment, a nucleic acid construct is provided comprising apromoter operably linked to an intron and 5′-UTR. In one embodiment theconstruct comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, or SEQ ID NO:39. In one embodiment, a nucleicacid construct is provided comprising a ubiquitin promoter sequencecomprising or consisting of a sequence at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical toSEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:39 operablylinked to a polylinker. Optionally, the construct can further comprise3′-UTR that is operably linked to the 3′ end of the polylinker. In anembodiment, a gene expression cassette is provided comprising aubiquitin promoter sequence wherein the promoter sequence comprises orconsists of a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, or SEQ ID NO:39. operably linked to anon-ubiquitin transgene. Optionally, the construct can further comprise3′-UTR that is operably linked to the 3′ end of the non-ubiquitintransgene. In one embodiment the 3′-UTR sequence consists of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36. In an illustrativeembodiment, the transgene can be an insecticidal resistance transgene,an herbicide tolerance transgene, a nitrogen use efficiency transgene, awater us efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof. In one embodiment the transgene is an herbicide resistancegene. In one embodiment a vector is provided comprising 1, 2, 3 or 4promoter sequences independently selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36.

In an embodiment, a gene expression cassette comprises a ubiquitinpromoter, a ubiquitin 5′-UTR, a ubiquitin intron, and a ubiquitin3′-UTR. In an embodiment, a ubiquitin promoter, a ubiquitin 5′-UTR, aubiquitin intron, and a ubiquitin 3′-UTR can each be independently aPanicum virgatum, Brachypodium distachyon or Setaria italica ubiquitinpromoter; Panicum virgatum Brachypodium distachyon or Setaria italicaubiquitin 5′-UTR; Panicum virgatum, Brachypodium distachyon or Setariaitalica ubiquitin intron; and, a Panicum virgatum, Brachypodiumdistachyon or Setaria italica ubiquitin 3′-UTR. In an embodiment, a geneexpression cassette comprises: a) a promoter, wherein the promoter is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, orSEQ ID NO:36; b) a 3′-UTR, wherein the 3′-UTR is at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100%identical to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:37; c)a 5′-UTR, wherein the 5′-UTR is at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38; or, d)an intron, wherein the intron is at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:39.

For example, a gene expression cassette may include both a promoter, anintron, and a 5′-UTR wherein the promoter is a polynucleotide of SEQ IDNO:1, the intron is a polynucleotide of SEQ ID NO:7, and the 5′-UTR is apolynucleotide of SEQ ID NO:11. Likewise, a gene expression cassette mayinclude both a promoter, an intron, and a 5′-UTR wherein the promoter isa polynucleotide of SEQ ID NO:2, the intron is a polynucleotide of SEQID NO:8, and the 5′-UTR is a polynucleotide of SEQ ID NO:12.Furthermore, a gene expression cassette may include both a promoter, anintron, and a 5′-UTR wherein the promoter is a polynucleotide of SEQ IDNO:3, the intron is a polynucleotide of SEQ ID NO:9 and/or SEQ ID NO:10,and the 5′-UTR is a polynucleotide of SEQ ID NO:13. In addition, a geneexpression cassette may include both a promoter, an intron, and a 5′-UTRwherein the promoter is a polynucleotide of SEQ ID NO:35, the intron isa polynucleotide of SEQ ID NO:37, and the 5′-UTR is a polynucleotide ofSEQ ID NO:38.

For example, a gene expression cassette may include both a promoter, anintron, a 5′-UTR, and a 3′-UTR wherein the promoter is a polynucleotideof SEQ ID NO:1, the intron is a polynucleotide of SEQ ID NO:7, the5′-UTR is a polynucleotide of SEQ ID NO:11, and the 3′-UTR is apolynucleotide of SEQ ID NO. 4. Likewise, a gene expression cassette mayinclude both a promoter, an intron, a 5′-UTR, and a 3′-UTR wherein thepromoter is a polynucleotide of SEQ ID NO:2, the intron is apolynucleotide of SEQ ID NO:8, the 5′-UTR is a polynucleotide of SEQ IDNO:12, and the 3′-UTR is a polynucleotide of SEQ ID NO:5. Furthermore, agene expression cassette may include both a promoter, an intron, a5′-UTR, and a 3′-UTR wherein the promoter is a polynucleotide of SEQ IDNO:3, the intron is a polynucleotide of SEQ ID NO:9 and/or SEQ ID NO:10,the 5′-UTR is a polynucleotide of SEQ ID NO:13 or 14, and the 3′-UTR isa polynucleotide of SEQ ID NO:6. In addition, a gene expression cassettemay include both a promoter, an intron, a 5′-UTR, and a 3′-UTR whereinthe promoter is a polynucleotide of SEQ ID NO:35, the intron is apolynucleotide of SEQ ID NO:37, the 5′-UTR is a polynucleotide of SEQ IDNO:38, and the 3′-UTR is a polynucleotide of SEQ ID NO:36.

In addition, a gene expression cassette may include both a promoter, anda 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:1 and a3′-UTR of SEQ ID NO:6. In an embodiment, a gene expression cassette mayinclude both a promoter and a 3′-UTR wherein the promoter is apolynucleotide of SEQ ID NO:1 and a 3′-UTR of SEQ ID NO:5. In anembodiment, a gene expression cassette may include both a promoter and a3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:1 and a3′-UTR of SEQ ID NO:4. In an embodiment, a gene expression cassette mayinclude both a promoter and a 3′-UTR wherein the promoter is apolynucleotide of SEQ ID NO:35 and a 3′-UTR of SEQ ID NO:36.

In an embodiment, a gene expression cassette comprises a ubiquitinpromoter, ubiquitin 5′-UTR, and a ubiquitin 3′-UTR that are operablylinked to a non-ubiquitin transgene. In an embodiment, a gene expressioncassette comprises a ubiquitin promoter, a ubiquitin intron, ubiquitin5′-UTR, and a ubiquitin 3′-UTR that are operably linked to anon-ubiquitin transgene.

A promoter, an intron, a 5′-UTR, and 3′-UTR can be operably linked todifferent transgenes within a gene expression cassette when a geneexpression cassette includes one or more transgenes. In an illustrativeembodiment, a gene expression cassette comprises a ubiquitin promoterthat is operably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water use efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof. In an illustrativeembodiment, a gene expression cassette comprises a ubiquitin promoter,an intron, and a 5′-UTR that are operably linked to a transgene, whereinthe transgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water useefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof. In anillustrative embodiment, a gene expression cassette comprises aubiquitin 3′-UTR that is operably linked to a transgene, wherein thetransgene encodes for a gene product that enhances insecticidalresistance, herbicide tolerance, nitrogen use efficiency, water usefficiency, nutritional quality or combinations thereof.

A ubiquitin intron and a 5′-UTR can be operably linked to differentpromoters within a gene expression cassette. In an illustrativeembodiment, the promoters originate from a plant (e.g., Zea maysubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic viruspromoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). Inan illustrative embodiment, a gene expression cassette comprises aubiquitin promoter that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water useefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof.

In an embodiment, a vector comprises a gene expression cassette asdisclosed herein. In an embodiment, a vector can be a plasmid, a cosmid,a bacterial artificial chromosome (BAC), a bacteriophage, a virus, or anexcised polynucleotide fragment for use in direct transformation or genetargeting such as a donor DNA.

In accordance with one embodiment a nucleic acid vector is providedcomprising a recombinant gene cassette wherein the recombinant genecassette comprises a ubiquitin based promoter operably linked to apolylinker sequence, a non-ubiquitin transgene or combination thereof.In one embodiment the recombinant gene cassette comprises a ubiquitinbased promoter operably linked to a non-ubiquitin transgene. In oneembodiment the recombinant gene cassette comprises a ubiquitin basedpromoter as disclosed herein operably linked to a polylinker sequence.The polylinker is operably linked to the ubiquitin based promoter in amanner such that insertion of a coding sequence into one of therestriction sites of the polylinker will operably link the codingsequence allowing for expression of the coding sequence when the vectoris transfected into a host cell.

In accordance with one embodiment the ubiquitin based promoter comprisesSEQ ID NO: 1 or a sequence that has 90, 95 or 99% sequence identity withSEQ ID NO: 1. In accordance with one embodiment the promoter sequencehas a total length of no more than 1.5, 2, 2.5, 3 or 4 kb. In accordancewith one embodiment the ubiquitin based promoter consists of SEQ ID NO:1 or a 1029 bp sequence that has 90, 95 or 99% sequence identity withSEQ ID NO: 1.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette that consists of SEQ ID NO: 15, anon-ubiquitin transgene and a 3′-UTR, wherein SEQ ID NO: 15 is operablylinked to the 5′ end of the non-ubiquitin transgene and the 3′-UTR isoperably linked to the 3′ end of the non-ubiquitin transgene. In afurther embodiment the 3′ untranslated sequence comprises SEQ ID NO: 4or a sequence that has 90, 95, 99 or 100% sequence identity with SEQ IDNO: 4. In accordance with one embodiment a nucleic acid vector isprovided comprising a gene cassette that consists of SEQ ID NO: 15, or a2077 bp sequence that has 90, 95, or 99% sequence identity with SEQ IDNO: 15, a non-ubiquitin transgene and a 3′-UTR, wherein SEQ ID NO: 15 isoperably linked to the 5′ end of the non-ubiquitin transgene and the3′-UTR is operably linked to the 3′ end of the non-ubiquitin transgene.In a further embodiment the 3′ untranslated sequence comprises SEQ IDNO: 4 or a sequence that has 90, 95, 99 or 100% sequence identity withSEQ ID NO: 4. In a further embodiment the 3′ untranslated sequenceconsists of SEQ ID NO: 4, or a 1026 bp sequence that has 90, 95, or 99%sequence identity with SEQ ID NO: 4.

In accordance with one embodiment the nucleic acid vector furthercomprises a sequence encoding a selectable maker. In accordance with oneembodiment the recombinant gene cassette is operably linked to anAgrobacterium T-DNA border. In accordance with one embodiment therecombinant gene cassette further comprises a first and second T-DNAborder, wherein first T-DNA border is operably linked to one end of thegene construct, and said second T-DNA border is operably linked to theother end of the gene construct. The first and second AgrobacteriumT-DNA borders can be independently selected from T-DNA border sequencesoriginating from bacterial strains selected from the group consisting ofa nopaline synthesizing Agrobacterium T-DNA border, an ocotopinesynthesizing Agrobacterium T-DNA border, a succinamopine synthesizingAgrobacterium T-DNA border, or any combination thereof. In oneembodiment an Agrobacterium strain selected from the group consisting ofa nopaline synthesizing strain, a mannopine synthesizing strain, asuccinamopine synthesizing strain, or an octopine synthesizing strain isprovided, wherein said strain comprises a plasmid wherein the plasmidcomprises a transgene operably linked to a sequence selected from SEQ IDNO: 1, SEQ ID NO: 15 or a sequence having 90, 95, or 99% sequenceidentity with SEQ ID NO: 1 or SEQ ID NO: 15.

Transgenes of interest and suitable for use in the present disclosedconstructs include, but are not limited to, coding sequences that confer(1) resistance to pests or disease, (2) resistance to herbicides, and(3) value added traits as disclosed in WO2013116700 (DGT-28),US20110107455 (DSM-2), U.S. Pat. No. 8,283,522 (AAD-12); U.S. Pat. No.7,838,733 (AAD-1); U.S. Pat. Nos. 5,188,960; 5,691,308; 6,096,708; and6,573,240 (Cry1F); U.S. Pat. Nos. 6,114,138; 5,710,020; and 6,251,656(Cry1Ac); U.S. Pat. Nos. 6,127,180; 6,624,145 and 6,340,593 (Cry34Ab1);U.S. Pat. Nos. 6,083,499; 6,548,291 and 6,340,593 (Cry35Ab1), thedisclosures of which are incorporated herein. In accordance with oneembodiment the transgene encodes a selectable marker or a gene productconferring insecticidal resistance, herbicide tolerance, nitrogen useefficiency, water use efficiency, or nutritional quality.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette wherein the gene cassette comprises apromoter region operably linked to the 5′ end of a transgene wherein the3′ end of the transgene is linked to a 3′ untranslated region. In oneembodiment the promoter region comprises SEQ ID NO: 1 or a sequence thathas 90, 95 or 99% sequence identity with SEQ ID NO: 1. In accordancewith one embodiment the promoter region consists of SEQ ID NO: 1 or SEQID NO: 15. In one embodiment the 3′ untranslated sequence comprises SEQID NO: 4 or a sequence that has 90, 95 or 99% sequence identity with SEQID NO: 4, and in one embodiment the 3′ untranslated sequence consists ofSEQ ID NO: 4 or a 1026 bp sequence having 90, 95 or 99% sequenceidentity with SEQ ID NO: 4.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette wherein the gene cassette comprises apromoter region operably linked to the 5′ end of a 5′ untranslatedsequence, wherein the 3′ end of the 5′ untranslated sequence is operablylinked to the 5′ end of the transgene wherein the 3′ end of thetransgene is linked to a 3′ untranslated region. In one embodiment thepromoter region comprises or consists of SEQ ID NO: 1 or a sequence thathas 90, 95 or 99% sequence identity with SEQ ID NO: 1. In one embodimentthe promoter region consists of SEQ ID NO: 1 or a 1029 bp sequence thathas 90, 95 or 99% sequence identity with SEQ ID NO: 1. In accordancewith one embodiment the 5′ untranslated sequence comprises or consistsof SEQ ID NO: 11 or a sequence that has 90% sequence identity with SEQID NO: 11. In accordance with one embodiment the 5′ untranslatedsequence consists of SEQ ID NO: 11 or a 55 bp sequence that has 90%sequence identity with SEQ ID NO: 11. In one embodiment the 3′untranslated sequence comprises or consists of SEQ ID NO: 4 or asequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 4. Inone embodiment the 3′ untranslated sequence consists of SEQ ID NO: 4 ora 1026 bp sequence that has 90, 95 or 99% sequence identity with SEQ IDNO: 4. In a further embodiment the nucleic acid vector further comprisesa ubiquitin intron inserted between the 5′ untranslated region and thetransgene, and operably linked to the promoter and transgene. In oneembodiment the ubiquitin intron comprises or consists of SEQ ID NO: 7 ora sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 7.In one embodiment the ubiquitin intron consists of SEQ ID NO: 7 or a 993bp sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 7.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette wherein the gene cassette comprises apromoter region operably linked to the 5′ end of a transgene wherein the3′ end of the transgene is linked to a 3′ untranslated region. In oneembodiment the promoter region comprises SEQ ID NO: 40 or a sequencethat has 90, 95 or 99% sequence identity with SEQ ID NO: 40.

(SEQ ID NO: 40) CTGCTCGTTCAGCCCACAGTAACACGCCGTGCGACATGCAGATGCCCTCCACCACGCCGACCAACCCCAAGTCCGCCGCGCTCGTCCACGGCGCCATCCGCATCCGCGCGTCAACGTCATCCGGAGGAGGCGAGCGCGATGTCGACGGCCACGGCGGCGGCGGACACGACGGCGACGCCCCGACTCCGCGCGCGCGTCAAGGCTGCAGTGGCGTCGTGGTGGCCGTCCGCCTGCACGAGATCCCCGCGTGGACGAGCGCCGCCTCCACCCAGCCCCTATATCGAGAAATCAACGGTGGGCTCGAGCTCCTCAGCAACCTCCCCACCCCCCCTTCCGACCACGCTCCCTTCCCCCGTGCCCCTCTTCTCCGTAAACCCGAGCCGCCGAGAACAACACCAACGAAAGGGCGAAGAGAATCGCCATAGAGAGGAGATGGGCGGAGGCGGATAGTTTCAGCCATTCACGGAGAAATGGGGAGGAGAGAACACGACATCATACGGACGCGACCCTCTAGCTGGCTGGCTGTCCTAAAGAATCGAACGGAATCGCTGCGCCAGGAGAAAACGAACGGTCCTGAAGCATGTGCGCCCGGTTCTTCCAAAACACTTATCTTTAAGATTGAAGTAGTATATATGACTGAAATTTTTACAAGGTTTTTCCCCATAAAACAGGTGAGCTTATCTCATCCTTTTGTTTAGGATGTACGTATTATATATGACTGAATATTTTTTATTTTCATTGAATGAAGATTTTCGACCCCCCAAAAATAAAAAACGGAGGGAGTACCTTTGTGCCGTGTATATGGACTAGAGCCATCGGGACGTTTCCGGAGACTGCGTGGTGGGGGCGATGGACGCACAACGACCGCATTTTCGGTTGCCGACTCGCCGTTCGCATCTGGTAGGCACGACTCGTCGGGTTCGGCTCTTGCGTGAGCCGTGACGTAACAGACCCGTTCTCTTCCCCCGTCTGGCCATCCATAAATCCCCCCTCCATCGGCTTCCCTTTCCTCAATCCAGCACCCTGATTCCGATCGAAAAGTCCCCGCAAGAGCAAGCGACCGATCTCGTGAATCTCCGTCAAGIn accordance with one embodiment the promoter region consists of SEQ IDNO: 40 or a 1084 bp sequence having 90, 95 or 99% sequence identity withSEQ ID NO: 40. In accordance with one embodiment the promoter regionconsists of SEQ ID NO: 40. In one embodiment the 3′ untranslatedsequence consists of SEQ ID NO: 4 or a 1026 bp sequence that has 90, 95or 99% sequence identity with SEQ ID NO: 4, and in one embodiment the 3′untranslated sequence consists of SEQ ID NO: 4.

In an embodiment, a cell or plant is provided comprising a geneexpression cassette as disclosed herein. In an embodiment, a cell orplant comprises a vector comprising a gene expression cassette asdisclosed herein. In an embodiment, a vector can be a plasmid, a cosmid,a bacterial artificial chromosome (BAC), a bacteriophage, or a virus.Thereby, a cell or plant comprising a gene expression cassette asdisclosed herein is a transgenic cell or transgenic plant, respectively.In an embodiment, a transgenic plant can be a monocotyledonous plant. Inan embodiment, a transgenic monocotyledonous plant can be, but is notlimited to maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane,and millet. In an embodiment, a transgenic plant can be a dicotyledonousplant. In an embodiment, a transgenic dicotyledonous plant can be, butis not limited to soybean, cotton, sunflower, and canola. An embodimentalso includes a transgenic seed from a transgenic plant as disclosedherein.

In an embodiment, a gene expression cassette includes two or moretransgenes. The two or more transgenes may not be operably linked to thesame promoter, intron, or 5′-UTR or 3′-UTR as disclosed herein. In anembodiment, a gene expression cassette includes one or more transgenes.In an embodiment with one or more transgenes, at least one transgene isoperably linked to a promoter, intron, 5′-UTR, or 3′-UTR or the subjectdisclosure.

Selectable Markers

Various selectable markers also described as reporter genes can beincorporated into a chosen expression vector to allow for identificationand selectable of transformed plants (“transformants”). Many methods areavailable to confirm expression of selectable markers in transformedplants, including for example DNA sequencing and PCR (polymerase chainreaction), Southern blotting, RNA blotting, immunological methods fordetection of a protein expressed from the vector, e.g., precipitatedprotein that mediates phosphinothricin resistance, or visual observationof other proteins such as reporter genes encoding β-glucuronidase (GUS),luciferase, green fluorescent protein (GFP), yellow fluorescent protein(YFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT),alkaline phosphatase, and the like (See Sambrook, et al., MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press,N.Y., 2001, the content of which is incorporated herein by reference inits entirety).

Selectable marker genes are utilized for selection of transformed cellsor tissues. Selectable marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II (NEO)and hygromycin phosphotransferase (HPT) as well as genes conferringresistance to herbicidal compounds. Herbicide resistance genes generallycode for a modified target protein insensitive to the herbicide or foran enzyme that degrades or detoxifies the herbicide in the plant beforeit can act. For example, resistance to glyphosate has been obtained byusing genes coding for mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Genes and mutantsfor EPSPS are well known, and further described below. Resistance toglufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D)have been obtained by using bacterial genes encoding pat or DSM-2, anitrilase, an aad-1, or an aad-12 gene, which detoxifies the respectiveherbicides.

In an embodiment, herbicides can inhibit the growing point or meristem,including imidazolinone or sulfonylurea, and genes forresistance/tolerance of acetohydroxyacid synthase (AHAS) andacetolactate synthase (ALS) for these herbicides are well known.Glyphosate resistance genes include mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and dgt-28 genes(via the introduction of recombinant nucleic acids and/or various formsof in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosateacetyl transferase (GAT) genes, respectively). Resistance genes forother phosphono compounds include bar genes from Streptomyces species,including Streptomyces hygroscopicus and Streptomyces viridichromogenes,and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). Exemplary genes conferring resistance tocyclohexanediones and/or aryloxyphenoxypropanoic acid (includingHaloxyfop, Diclofop, Fenoxyprop, Fluazifop, Quizalofop) include genes ofacetyl coenzyme A carboxylase (ACCase)-Acc1-S1, Acc1-S2 and Acc1-S3. Inan embodiment, herbicides can inhibit photosynthesis, including triazine(psbA and 1s+ genes) or benzonitrile (nitrilase gene).

In an embodiment, selectable marker genes include, but are not limitedto genes encoding: neomycin phosphotransferase II; cyanamide hydratase;aspartate kinase; dihydrodipicolinate synthase; tryptophandecarboxylase; dihydrodipicolinate synthase and desensitized aspartatekinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase(NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolatereductase (DHFR); phosphinothricin acetyltransferase;2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase;5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase;acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32kD photosystem II polypeptide (psbA).

An embodiment also includes genes encoding resistance to:chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil;glyphosate; and phosphinothricin.

The above list of selectable marker genes is not meant to be limiting.Any reporter or selectable marker gene are encompassed by the presentinvention.

Selectable marker genes are synthesized for optimal expression in aplant. For example, in an embodiment, a coding sequence of a gene hasbeen modified by codon optimization to enhance expression in plants. Aselectable marker gene can be optimized for expression in a particularplant species or alternatively can be modified for optimal expression indicotyledonous or monocotyledonous plants. Plant preferred codons may bedetermined from the codons of highest frequency in the proteinsexpressed in the largest amount in the particular plant species ofinterest. In an embodiment, a selectable marker gene is designed to beexpressed in plants at a higher level resulting in higher transformationefficiency. Methods for plant optimization of genes are well known.Guidance regarding the optimization and production of synthetic DNAsequences can be found in, for example, WO2013016546, WO2011146524,WO1997013402, U.S. Pat. No. 6,166,302, and U.S. Pat. No. 5,380,831,herein incorporated by reference.

Transformation

Suitable methods for transformation of plants include any method bywhich DNA can be introduced into a cell, for example and withoutlimitation: electroporation (see, e.g., U.S. Pat. No. 5,384,253);micro-projectile bombardment (see, e.g., U.S. Pat. Nos. 5,015,580,5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865);Agrobacterium-mediated transformation (see, e.g., U.S. Pat. Nos.5,635,055, 5,824,877, 5,591,616; 5,981,840, and 6,384,301); andprotoplast transformation (see, e.g., U.S. Pat. No. 5,508,184).

A DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as agitation with silicon carbidefibers (See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), or the DNAconstructs can be introduced directly to plant tissue using biolisticmethods, such as DNA particle bombardment (see, e.g., Klein et al.(1987) Nature 327:70-73). Alternatively, the DNA construct can beintroduced into the plant cell via nanoparticle transformation (see,e.g., US Patent Publication No. 20090104700, which is incorporatedherein by reference in its entirety).

In addition, gene transfer may be achieved using non-Agrobacteriumbacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virusand cassava vein mosaic virus and/or tobacco mosaic virus, See, e.g.,Chung et al. (2006) Trends Plant Sci. 11(1):1-4.

Through the application of transformation techniques, cells of virtuallyany plant species may be stably transformed, and these cells may bedeveloped into transgenic plants by well-known techniques. For example,techniques that may be particularly useful in the context of cottontransformation are described in U.S. Pat. Nos. 5,846,797, 5,159,135,5,004,863, and 6,624,344; techniques for transforming Brassica plants inparticular are described, for example, in U.S. Pat. No. 5,750,871;techniques for transforming soy bean are described, for example, in U.S.Pat. No. 6,384,301; and techniques for transforming maize are described,for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616, andInternational PCT Publication WO 95/06722.

After effecting delivery of an exogenous nucleic acid to a recipientcell, a transformed cell is generally identified for further culturingand plant regeneration. In order to improve the ability to identifytransformants, one may desire to employ a selectable marker gene withthe transformation vector used to generate the transformant. In anillustrative embodiment, a transformed cell population can be assayed byexposing the cells to a selective agent or agents, or the cells can bescreened for the desired marker gene trait.

Cells that survive exposure to a selective agent, or cells that havebeen scored positive in a screening assay, may be cultured in media thatsupports regeneration of plants. In an embodiment, any suitable planttissue culture media may be modified by including further substances,such as growth regulators. Tissue may be maintained on a basic mediawith growth regulators until sufficient tissue is available to beginplant regeneration efforts, or following repeated rounds of manualselection, until the morphology of the tissue is suitable forregeneration (e.g., at least 2 weeks), then transferred to mediaconducive to shoot formation. Cultures are transferred periodicallyuntil sufficient shoot formation has occurred. Once shoots are formed,they are transferred to media conducive to root formation. Oncesufficient roots are formed, plants can be transferred to soil forfurther growth and maturity.

To confirm the presence of a desired nucleic acid comprising constructsprovided in regenerating plants, a variety of assays may be performed.Such assays may include: molecular biological assays, such as Southernand northern blotting and PCR; biochemical assays, such as detecting thepresence of a protein product, e.g., by immunological means (ELISA,western blots, and/or LC-MS MS spectrophotometry) or by enzymaticfunction; plant part assays, such as leaf or root assays; and/oranalysis of the phenotype of the whole regenerated plant.

Transgenic events may be screened, for example, by PCR amplificationusing, e.g., oligonucleotide primers specific for nucleic acid moleculesof interest. PCR genotyping is understood to include, but not be limitedto, polymerase-chain reaction (PCR) amplification of genomic DNA derivedfrom isolated host plant callus tissue predicted to contain a nucleicacid molecule of interest integrated into the genome, followed bystandard cloning and sequence analysis of PCR amplification products.Methods of PCR genotyping have been well described (see, e.g., Rios etal. (2002) Plant J. 32:243-53), and may be applied to genomic DNAderived from any plant species or tissue type, including cell cultures.Combinations of oligonucleotide primers that bind to both targetsequence and introduced sequence may be used sequentially or multiplexedin PCR amplification reactions. Oligonucleotide primers designed toanneal to the target site, introduced nucleic acid sequences, and/orcombinations of the two may be produced. Thus, PCR genotyping strategiesmay include, for example and without limitation: amplification ofspecific sequences in the plant genome; amplification of multiplespecific sequences in the plant genome; amplification of non-specificsequences in the plant genome; and combinations of any of the foregoing.One skilled in the art may devise additional combinations of primers andamplification reactions to interrogate the genome. For example, a set offorward and reverse oligonucleotide primers may be designed to anneal tonucleic acid sequence(s) specific for the target outside the boundariesof the introduced nucleic acid sequence.

Forward and reverse oligonucleotide primers may be designed to annealspecifically to an introduced nucleic acid molecule, for example, at asequence corresponding to a coding region within a nucleotide sequenceof interest comprised therein, or other parts of the nucleic acidmolecule. Primers may be used in conjunction with primers describedherein. Oligonucleotide primers may be synthesized according to adesired sequence and are commercially available (e.g., from IntegratedDNA Technologies, Inc., Coralville, Iowa). Amplification may be followedby cloning and sequencing, or by direct sequence analysis ofamplification products. In an embodiment, oligonucleotide primersspecific for the gene target are employed in PCR amplifications.

Method of Expressing a Transgene

In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a ubiquitin promoter operablylinked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant comprising growing a plantcomprising a ubiquitin 5′-UTR operably linked to at least one transgene.In an embodiment, a method of expressing at least one transgene in aplant comprising growing a plant comprising a ubiquitin intron operablylinked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant comprising growing a plantcomprising a ubiquitin promoter, a ubiquitin 5′-UTR, and a ubiquitinintron operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant comprisinggrowing a plant comprising a ubiquitin 3′-UTR operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene in a plant tissue or plant cell comprising culturing aplant tissue or plant cell comprising a ubiquitin promoter operablylinked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant tissue or plant cellcomprising culturing a plant tissue or plant cell comprising a ubiquitin5′-UTR operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant tissue or plantcell comprising culturing a plant tissue or plant cell comprising aubiquitin intron operably linked to at least one transgene. In anembodiment, a method of expressing at least one transgene in a planttissue or plant cell comprising culturing a plant tissue or plant cellcomprising a ubiquitin promoter, a ubiquitin 5′-UTR, and a ubiquitinintron operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant tissue or plantcell comprising culturing a plant tissue or plant cell comprising aubiquitin 3′-UTR operably linked to at least one transgene.

In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a gene expression cassettecomprising a ubiquitin promoter operably linked to at least onetransgene. In one embodiment the ubiquitin promoter consists of asequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:35, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:39 or asequence that has 90, 95 or 995 sequence identity with a sequenceselected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:35, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:39. In anembodiment, a method of expressing at least one transgene in a plantcomprises growing a plant comprising a gene expression cassettecomprising a ubiquitin intron operably linked to at least one transgene.In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a gene expression cassettecomprising a ubiquitin 5′-UTR operably linked to at least one transgene.In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a gene expression cassettecomprising a ubiquitin promoter, a ubiquitin 5′-UTR, and a ubiquitinintron operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant comprises growinga plant comprising a gene expression cassette comprising a ubiquitin3′-UTR operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette a ubiquitin promoter operably linked to at least onetransgene. In an embodiment, a method of expressing at least onetransgene in a plant tissue or plant cell comprises culturing a planttissue or plant cell comprising a gene expression cassette a ubiquitinintron operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette a ubiquitin 5′-UTR operably linked to at least onetransgene. In an embodiment, a method of expressing at least onetransgene in a plant tissue or plant cell comprises culturing a planttissue or plant cell comprising a gene expression cassette a ubiquitinpromoter, a ubiquitin 5′-UTR, and a ubiquitin intron operably linked toat least one transgene. In an embodiment, a method of expressing atleast one transgene in a plant tissue or plant cell comprises culturinga plant tissue or plant cell comprising a gene expression cassettecomprising a ubiquitin 3′-UTR operably linked to at least one transgene.

Transgenic Plants

In an embodiment, a plant, plant tissue, or plant cell comprises aubiquitin promoter. In an embodiment, a ubiquitin promoter can be aPanicum virgatum, Brachypodium distachyon or Setaria italica ubiquitinpromoter. In an embodiment, a plant, plant tissue, or plant cellcomprises a gene expression cassette comprises a promoter, wherein thepromoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 or SEQ ID NO:35 wherein the promoter is operably linked to anon-ubiquitin transgene. In an embodiment, a plant, plant tissue, orplant cell comprises a gene expression cassette comprising a sequenceselected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:35, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:39 or a sequencethat has 90, 95 or 995 sequence identity with a sequence selected fromSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, and SEQ ID NO:39 that is operably linked to anon-ubiquitin transgene. In an illustrative embodiment, a plant, planttissue, or plant cell comprises a gene expression cassette comprising aubiquitin promoter that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water usefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a 3′-UTR. In an embodiment, a plant,plant tissue, or plant cell comprises a gene expression cassettecomprising a ubiquitin 3′-UTR. In an embodiment, the ubiquitin 3′-UTR isa Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin3′-UTR. In an embodiment, a 3′-UTR can be the Brachypodium distachyonubiquitin1 C (Ubi1C) 3′-UTR, Brachypodium distachyon ubiquitin1 3′-UTR,or Setaria italica ubiquitin 3′-UTR.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising an intron, wherein the intron is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, or SEQ ID NO:37. In an embodiment, a gene expression cassettecomprises a ubiquitin intron that is operably linked to a promoter,wherein the promoter is a Panicum virgatum, Brachypodium distachyon orSetaria italica ubiquitin promoter, or a promoter that originates from aplant (e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava veinmosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciensdelta mas). In an embodiment, a plant, plant tissue, or plant cellcomprises a gene expression cassette comprising a ubiquitin intron thatis operably linked to a transgene. In an illustrative embodiment, aplant, plant tissue, or plant cell comprising a gene expression cassettecomprising a ubiquitin intron that is operably linked to a transgene,wherein the transgene can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater us efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a 5′-UTR, wherein the 5′-UTR is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:14, or SEQ ID NO:38. In an embodiment, a gene expressioncassette comprises a ubiquitin intron that is operably linked to apromoter, wherein the promoter is a Panicum virgatum, Brachypodiumdistachyon or Setaria italica ubiquitin promoter, or a promoter thatoriginates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus(e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g.,Agrobacterium tumefaciens delta mas). In an embodiment, a plant, planttissue, or plant cell comprises a gene expression cassette comprising aubiquitin 5′-UTR that is operably linked to a transgene. In anillustrative embodiment, a plant, plant tissue, or plant cell comprisinga gene expression cassette comprising a ubiquitin 5′-UTR that isoperably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water us efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a ubiquitin promoter and a ubiquitin3′-UTR. In an embodiment, a plant, plant tissue, or plant cell comprisesa ubiquitin promoter and 3′-UTR can each be independently a Panicumvirgatum, Brachypodium distachyon or Setaria italica ubiquitin promoterand a Panicum virgatum, Brachypodium distachyon or Setaria italicaubiquitin promoter. In an embodiment, a plant, plant tissue, or plantcell comprises a gene expression cassette comprising a) a promoter,wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, or SEQ ID NO:35 and b) a 3′-UTR, wherein the3′-UTR is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, or SEQ ID NO:36.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a ubiquitin promoter, ubiquitin 5′-UTR,ubiquitin intron, and a ubiquitin 3′-UTR that are operably linked to atransgene. The promoter, intron, 5′-UTR, and 3′-UTR can be operablylinked to different transgenes within a gene expression cassette when agene expression cassette includes two or more transgenes. In anillustrative embodiment, a gene expression cassette comprises aubiquitin promoter that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water usefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof. In anillustrative embodiment, a gene expression cassette comprises aubiquitin intron that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water usefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof. In anembodiment, a gene expression cassette comprises a ubiquitin intron thatis operably linked to a promoter, wherein the promoter is a Panicumvirgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter,or a promoter that originates from a plant (e.g., Zea mays ubiquitin 1promoter), a virus (e.g., Cassava vein mosaic virus promoter) or abacteria (e.g., Agrobacterium tumefaciens delta mas). In an illustrativeembodiment, a gene expression cassette comprises a ubiquitin 5′-UTR thatis operably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water us efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof. In an embodiment, a geneexpression cassette comprises a ubiquitin 5′-UTR that is operably linkedto a promoter, wherein the promoter is a Panicum virgatum, Brachypodiumdistachyon or Setaria italica ubiquitin promoter, or a promoter thatoriginates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus(e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g.,Agrobacterium tumefaciens delta mas). In an illustrative embodiment, agene expression cassette comprises a ubiquitin 3′-UTR that is operablylinked to a transgene, wherein the 3′-UTR can be an insecticidalresistance transgene, an herbicide tolerance transgene, a nitrogen useefficiency transgene, a water us efficiency transgene, a nutritionalquality transgene, a DNA binding transgene, a selectable markertransgene, or combinations thereof.

In an embodiment, a plant, plant tissue, or plant cell comprises avector comprising a ubiquitin promoter, 5′-UTR, intron, and/or 3′-UTR asdisclosed herein. In an embodiment, a plant, plant tissue, or plant cellcomprises a vector comprising a ubiquitin promoter, 5′-UTR, intron,and/or 3′-UTR as disclosed herein operably linked to a non-ubiquitintransgene. In an embodiment, a plant, plant tissue, or plant cellcomprises a vector comprising a gene expression cassette as disclosedherein. In an embodiment, a vector can be a plasmid, a cosmid, abacterial artificial chromosome (BAC), a bacteriophage, or a virus.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises anon-endogenous ubiquitin derived promoter sequence operably linked to atransgene, wherein the ubiquitin derived promoter sequence comprises asequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35 or asequence having 90. 95, 98 or 99% sequence identity with SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35. In one embodiment a plant,plant tissue, or plant cell is provided wherein the plant, plant tissue,or plant cell comprises SEQ ID NO: 1, or a sequence that has 90%sequence identity with SEQ ID NO: 1 operably linked to a non-ubiquitintransgene. In one embodiment the plant, plant tissue, or plant cell is adicotyledonous or monocotyledonous plant or a cell or tissue derivedfrom a dicotyledonous or monocotyledonous plant. In one embodiment theplant is selected from the group consisting of maize, wheat, rice,sorghum, oats, rye, bananas, sugar cane, soybean, cotton, sunflower, andcanola. In one embodiment the plant is Zea mays. In accordance with oneembodiment the plant, plant tissue, or plant cell comprises SEQ ID NO:1, SEQ ID NO: 15 or a sequence having 90. 95, 98 or 99% sequenceidentity with SEQ ID NO: 1 or SEQ ID NO: 15 operably linked to anon-ubiquitin transgene. In one embodiment the plant, plant tissue, orplant cell comprises a promoter operably linked to a transgene whereinthe promoter consists of SEQ ID NO: 1, SEQ ID NO: 15 or a sequencehaving 90. 95, 98 or 99% sequence identity with SEQ ID NO: 1 or SEQ IDNO: 15. In accordance with one embodiment the gene construct comprisingnon-endogenous ubiquitin derived promoter sequence operably linked to atransgene is incorporated into the genome of the plant, plant tissue, orplant cell.

In one embodiment a non-Brachypodium plant, plant tissue, or plant cellis provided comprising SEQ ID NO: 1, or a sequence that has 90, 95, 98or 99% sequence identity with SEQ ID NO: 1, operably linked to atransgene. In accordance with one embodiment the non-Brachypodium plant,plant tissue, or plant cell is a dicotyledonous or monocotyledonousplant or plant cell or tissue derived from a dicotyledonous ormonocotyledonous plant. In one embodiment the plant is selected from thegroup consisting of maize, wheat, rice, sorghum, oats, rye, bananas,sugar cane, soybean, cotton, sunflower, and canola. In one embodimentthe plant is Zea mays. In accordance with one embodiment the promotersequence operably linked to a transgene is incorporated into the genomeof the plant, plant tissue, or plant cell. In one embodiment the plant,plant tissue, or plant cell further comprises a 5′ untranslated sequencecomprising SEQ ID NO: 11 or a sequence that has 90% sequence identitywith SEQ ID NO: 11, wherein the 5′ untranslated sequence is insertedbetween, and operably linked to, said promoter and said transgene. In afurther embodiment the plant, plant tissue, or plant cell furthercomprises an intron sequence inserted after the 5′ untranslatedsequence. In one embodiment the intron sequence is an intron sequenceisolated from a ubiquitin gene of Panicum virgatum, Brachypodiumdistachyon, or Setaria italica. In one embodiment the sequence comprisesor consists of SEQ ID NO: 7.

In one embodiment a non-Brachypodium plant, plant tissue, or plant cellis provided that comprises SEQ ID NO: 1, or a sequence that has 90. 95,98 or 99% sequence identity with SEQ ID NO: 1, operably linked to the 5′end of a transgene and a 3′ untranslated sequence comprising SEQ ID NO:4 or a sequence that has 90% sequence identity with SEQ ID NO: 4,wherein the 3′ untranslated sequence is operably linked to saidtransgene. In accordance with one embodiment the non-Brachypodium plant,plant tissue, or plant cell is a dicotyledonous or monocotyledonousplant or is a plant issue or cell derived from a dicotyledonous ormonocotyledonous plant. In one embodiment the plant is selected from thegroup consisting of maize, wheat, rice, sorghum, oats, rye, bananas,sugar cane, soybean, cotton, sunflower, and canola. In one embodimentthe plant is Zea mays. In accordance with one embodiment the promotersequence operably linked to a transgene is incorporated into the genomeof the plant, plant tissue, or plant cell. In one embodiment the plant,plant tissue, or plant cell further comprises a 5′ untranslated sequencecomprising SEQ ID NO: 11 or a sequence that has 90% sequence identitywith SEQ ID NO: 11, wherein the 5′ untranslated sequence is insertedbetween, and operably linked to, said promoter and said transgene. In afurther embodiment the plant, plant tissue, or plant cell furthercomprises an intron sequence inserted after the 5′ untranslatedsequence. In one embodiment the intron sequence is an intron sequenceisolated from a ubiquitin gene of Panicum virgatum, Brachypodiumdistachyon, or Setaria italica. In one embodiment the 5′ untranslatedsequence consists of SEQ ID NO: 11.

In one embodiment a non-Brachypodium plant, plant tissue, or plant cellis provided that comprises SEQ ID NO: 15, or a sequence having 90%sequence identity with SEQ ID NO: 15 operably linked to a transgene. Inone embodiment a non-Brachypodium plant, plant tissue, or plant cell isprovided that comprises a promoter operably linked to a transgene,wherein the promoter consists of SEQ ID NO: 15, or a sequence having 90%sequence identity with SEQ ID NO: 15. In a further embodimentnon-Brachypodium plant, plant tissue, or plant cell further comprises a3′ untranslated sequence of a ubiquitin gene of Panicum virgatum,Brachypodium distachyon, or Setaria italica. In one embodiment the 3′untranslated sequence comprises or consists of SEQ ID NO: 4 or asequence that has 90% sequence identity with SEQ ID NO: 4, wherein the3′ untranslated sequence is operably linked to 3′ end of the transgene.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be a monocotyledonous plant. Themonocotyledonous plant, plant tissue, or plant cell can be, but notlimited to corn, rice, wheat, sugarcane, barley, rye, sorghum, orchids,bamboo, banana, cattails, lilies, oat, onion, millet, and triticale.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be a dicotyledonous plant. Thedicotyledonous plant, plant tissue, or plant cell can be, but notlimited to rapeseed, canola, indian mustard, ethiopian mustard, soybean,sunflower, and cotton.

With regard to the production of genetically modified plants, methodsfor the genetic engineering of plants are well known in the art. Forinstance, numerous methods for plant transformation have been developed,including biological and physical transformation protocols fordicotyledonous plants as well as monocotyledonous plants (e.g.,Goto-Fumiyuki et al., Nature Biotech 17:282-286 (1999); Miki et al.,Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. andThompson, J. E. Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993)). Inaddition, vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available, for example, inGruber et al., Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds., CRC Press, Inc., Boca Raton, pp.89-119 (1993).

One of skill in the art will recognize that after the exogenous sequenceis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection can be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed cells can also be identified by screening for theactivities of any visible marker genes (e.g., the yfp, gfp,β-glucuronidase, luciferase, B or C1 genes) that may be present on therecombinant nucleic acid constructs. Such selection and screeningmethodologies are well known to those skilled in the art.

Physical and biochemical methods also may be used to identify plant orplant cell transformants containing inserted gene constructs. Thesemethods include but are not limited to: 1) Southern analysis or PCRamplification for detecting and determining the structure of therecombinant DNA insert; 2) Northern blot, S1 RNase protection,primer-extension or reverse transcriptase-PCR amplification fordetecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) Next GenerationSequencing analysis; 5) protein gel electrophoresis, Western blottechniques, immunoprecipitation, or enzyme-linked immunoassays (ELISA),where the gene construct products are proteins. Additional techniques,such as in situ hybridization, enzyme staining, and immunostaining, alsomay be used to detect the presence or expression of the recombinantconstruct in specific plant organs and tissues. The methods for doingall these assays are well known to those skilled in the art.

Effects of gene manipulation using the methods disclosed herein can beobserved by, for example, northern blots of the RNA (e.g., mRNA)isolated from the tissues of interest. Typically, if the mRNA is presentor the amount of mRNA has increased, it can be assumed that thecorresponding transgene is being expressed. Other methods of measuringgene and/or encoded polypeptide activity can be used. Different types ofenzymatic assays can be used, depending on the substrate used and themethod of detecting the increase or decrease of a reaction product orby-product. In addition, the levels of polypeptide expressed can bemeasured immunochemically, i.e., ELISA, RIA, EIA and other antibodybased assays well known to those of skill in the art, such as byelectrophoretic detection assays (either with staining or westernblotting). As one non-limiting example, the detection of the AAD-1(aryloxyalkanoate dioxygenase; see WO 2005/107437) and PAT(phosphinothricin-N-acetyltransferase), EC 2.3.1.183) proteins using anELISA assay is described in U.S. Patent Publication No. 20090093366which is herein incorporated by reference in its entirety. The transgenemay be selectively expressed in some cell types or tissues of the plantor at some developmental stages, or the transgene may be expressed insubstantially all plant tissues, substantially along its entire lifecycle. However, any combinatorial expression mode is also applicable.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above wherein the seed has the transgene or gene construct.The present disclosure further encompasses the progeny, clones, celllines or cells of the transgenic plants described above wherein saidprogeny, clone, cell line or cell has the transgene or gene construct.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

Example 1

Transformation of Agrobacterium tumefaciens

The binary expression vectors were transformed into Agrobacteriumtumefaciens strain DAt13192 (RecA minus ternary strain) (Int'l. Pat.Pub. No. WO2012016222). Bacterial colonies were isolated, and binaryplasmid DNA was isolated and confirmed via restriction enzyme digestion.

Corn Transformation

Agrobacterium Culture Initiation.

Agrobacterium cultures were streaked from glycerol stocks ontoAgrobacterium (AB) minimal medium (as disclosed in WO 2013090734, thedisclosure of which is incorporated herein by reference) and incubatedat 20° C. in the dark for 3 days. Agrobacterium cultures were thenstreaked onto a plate of YEP (see WO 2013090734) medium and incubated at20° C. in the dark for 1 day.

On the day of the experiment, a mixture of inoculation medium (see WO2013090734) and acetosyringone were prepared in a volume appropriate tothe number of bacterial strains comprising plant transformationconstructs in the experiment. Inoculation medium was pipetted into asterile, disposable 250 ml flask. Next, a 1 M stock solution ofacetosyringone in 100% dimethyl sulfoxide was added to the flaskcontaining inoculation medium in a volume appropriate to make a finalacetosyringone concentration of 200 μM. The required volumes ofInoculation medium and 1 M acetosyringone stock solution are listed inTABLE 1.

TABLE 1 The amount of inoculation medium/acetosyringone mixture to makeaccording to the number of constructs being prepared Number ofInoculation 1M aceto- constructs medium syringone to prepare (mL) stock(μL) 1 50 10 2 100 20 3 150 30 4 200 40 5 250 50

For each construct, 1-2 loops of Agrobacterium from the YEP plate weresuspended in 15 ml of the inoculation medium/acetosyringone mixtureinside a sterile, disposable 50 ml centrifuge tube, and the opticaldensity of the solution at 600 nm (OD₆₀₀) was measured in aspectrophotometer. The suspension was then diluted down to 0.25-0.35OD₆₀₀ using additional inoculation medium/acetosyringone mixture. Thetube of Agrobacterium suspension was then placed horizontally on aplatform shaker set at about 75 rpm at room temperature and incubatedbetween 1 and 4 hours before use.

Ear Sterilization and Embryo Isolation.

Ears from Zea mays cultivar B104 were harvested 10-12 days postpollination. Harvested ears were de-husked and surface-sterilized byimmersion in a 20% solution of commercial bleach (Ultra Clorox®Germicidal Bleach, 6.15% sodium hypochlorite) and two drops of Tween®20, for 20 minutes, followed by three rinses in sterile, deionized waterinside a laminar flow hood. Immature zygotic embryos (1.8-2.2 mm long)were aseptically excised from each ear and distributed into one or moremicro-centrifuge tubes containing 2.0 ml of Agrobacterium suspensioninto which 2 μl of 10% Break-Thru® S233 surfactant had been added.

Agrobacterium Co-Cultivation.

Upon completion of the embryo isolation activity, the tube of embryoswas closed and placed on a rocker platform for 5 minutes. The contentsof the tube were then poured out onto a plate of co-cultivation medium,and the liquid Agrobacterium suspension was removed with a sterile,disposable transfer pipette. The co-cultivation plate containing embryoswas placed at the back of the laminar flow hood with the lid ajar for 30minutes; after which time the embryos were oriented with the scutellumfacing up using a microscope. The co-cultivation plate with embryos wasthen returned to the back of the laminar flow hood with the lid ajar fora further 15 minutes. The plate was then closed, sealed with 3M®Micropore® tape, and placed in an incubator at 25° C. with 24 hours/daylight at approximately 60 μmol m⁻² s⁻¹ light intensity

Callus Selection and Regeneration of Transgenic Events.

Following the co-cultivation period, embryos were transferred to Restingmedium (see WO 2013090734). No more than 36 embryos were moved to eachplate. The plates were placed in clear boxes and incubated at 27° C.with 24 hours/day light at approximately 50 μmol m⁻² s⁻¹ light intensityfor 7-10 days. Callused embryos were then transferred onto Selection Imedium (see WO 2013090734). No more than 18 callused embryos were movedto each plate of Selection I. The plates were placed in clear boxes andincubated at 27° C. with 24 hours/day light at approximately 50 μmol m⁻²s⁻¹ light intensity for 7 days. Callused embryos were then transferredto Selection II medium (see WO 2013090734). No more than 12 callusedembryos were moved to each plate of Selection II media. The plates wereplaced in clear boxes and incubated at 27° C. with 24 hours/day light atapproximately 50 μmol m⁻² s⁻¹ light intensity for 14 days.

At this stage resistant calli were moved to Pre-Regeneration medium (seeWO 2013090734). No more than 9 calli were moved to each plate ofPre-Regeneration media. The plates were placed in clear boxes andincubated at 27° C. with 24 hours/day light at approximately 50 μmol m⁻²s⁻¹ light intensity for 7 days. Regenerating calli were then transferredto Regeneration medium in Phytatrays™ (see WO 2013090734). and incubatedat 28° C. with 16 hours light/8 hours dark per day at approximately 150μmol m⁻² s⁻¹ light intensity for 7-14 days or until shoots develop. Nomore than 5 calli were placed in each Phytatray™. Small shoots withprimary roots were then isolated and transferred to Shoot Elongationmedium (see WO 2013090734). Rooted plantlets about 6 cm or taller weretransplanted into soil and moved out to a growth chamber for hardeningoff.

YFP Transient Expression.

Transient YFP expression was observed in transformed embryos and after 3days of co-cultivation with Agrobacterium. The embryos were observedunder a stereomicroscope (Leica Microsystems, Buffalo Grove, Ill.) usinga YFP filter and 500 nm light source.

Transfer and Establishment of T₀ Plants in the Greenhouse.

Transgenic plants were transferred on a regular basis to the greenhouse.Plants were transplanted from Phytatrays™ to small pots (T. O. Plastics,3.5″ SVD, 700022C) filled with growing media (Premier Tech Horticulture,ProMix BX, 0581 P) and covered with humidomes to help acclimate theplants. Plants were placed in a Conviron growth chamber (28° C./24° C.,16-hour photoperiod, 50-70% RH, 200 μmol m⁻² s⁻¹ light intensity) untilreaching V3-V4 stage. This aided in acclimating the plants to soil andharsher temperatures. Plants were then moved to the greenhouse (LightExposure Type: Photo or Assimilation; High Light Limit: 1200 μmol m⁻²s⁻¹ photosynthetically active radiation (PAR); 16-hour day length; 27°C. Day/24° C. Night) and transplanted from the small pots to 5.5 inchpots. Approximately 1-2 weeks after transplanting to larger pots plantswere sampled for bioassay. One plant per event was assayed.

Example 2: Identification of the Promoters

The maize ubiquitin coding sequence was BLASTx searched in the Phytozome(Goodstein et al., 2012) database using Brachypodium distachyon andSetaria italica as target genomes.

Maize Ubiquitin (ZM Ubi1) Coding Sequence (SEQ ID NO: 18) ATGCAGATCTTTGTGAAAACCCTGACTGGCAAGACTATCACCCTCGAGGTGGAGTCGTCTGACACCATTGACAACGTTAAGGCCAAGATCCAGGACAAGGAGGGCATCCCCCCAGACCAGCAGCGGCTCATCTTTGCTGGCAAACAGCTTGAGGACGGGCGCACGCTTGCTGACTACAACATCCAGAAGGAGAGCACCCTCCACCTTGTGCTCCGTCTCAGGGGAGGCATGCAGATCTTTGTGAAAACCCTGACCGGCAAGACTATCACCCTCGAGGTGGAGTCCTCTGACACCATTGACAACGTCAAGGCCAAGATCCAGGACAAGGAGGGCATCCCTCCAGACCAGCAGCGGCTCATCTTTGCTGGGAAGCAGCTTGAGGACGGGCGCACGCTTGCCGACTACAACATCCAGAAGGAGAGCACCCTCCACTTGGTGCTGCGCCTCAGGGGAGGCATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACTATCACCCTCGAGGTGGAGTCTTCAGACACCATCGACAACGTCAAGGCCAAGATCCAGGACAAGGAGGGCATTCCCCCAGACCAGCAGCGGCTCATCTTTGCTGGAAAGCAGCTTGAGGACGGGCGCACGCTTGCCGACTACAACATCCAGAAGGAGAGCACCCTCCACTTGGTGCTGCGCCTCAGGGGAGGCATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACTATCACCCTCGAGGTGGAGTCTTCAGACACCATCGACAATGTCAAGGCCAAGATCCAGGACAAGGAGGGCATCCCACCGGACCAGCAGCGTTTGATCTTCGCTGGCAAGCAGCTGGAGGATGGCCGCACCCTTGCGGATTACAACATCCAGAAGGAGAGCACCCTCCACCTGGTGCTCCGTCTCAGGGGTGGTATGCAGATCTTTGTGAAGACACTCACTGGCAAGACAATCACCCTTGAGGTGGAGTCTTCGGATACCATTGACAATGTCAAGGCCAAGATCCAGGACAAGGAGGGCATCCCACCCGACCAGCAGCGCCTCATCTTCGCCGGCAAGCAGCTGGAGGATGGCCGCACCCTGGCGGATTACAACATCCAGAAGGAGAGCACTCTCCACCTGGTGCTCCGCCTCAGGGGTGGCATGCAGATTTTTGTGAAGACATTGACTGGCAAGACCATCACCTTGGAGGTGGAGAGCTCTGACACCATTGACAATGTGAAGGCCAAGATCCAGGACAAGGAGGGCATTCCCCCAGACCAGCAGCGTCTGATCTTTGCGGGCAAGCAGCTGGAGGATGGCCGCACTCTCGCGGACTACAACATCCAGAAGGAGAGCACCCTTCACCTTGTTCTCCGCCTCAGAGGTGGTATGCAGATCTTTGTAAAGACCCTGACTGGAAAAACCATAACCCTGGAGGTTGAGAGCTCGGACACCATCGACAATGTGAAGGCGAAGATCCAGGACAAGGAGGGCATCCCCCCGGACCAGCAGCGTCTGATCTTCGCCGGCAAACAGCTGGAGGATGGCCGCACCCTAGCAGACTACAACATCCAAAAGGAGAGCACCCTCCACCTTGTGCTCCGTCTCCGTGGTGGTCAG TAA

The protein alignments are shown in FIG. 1. Two sequences that alignedwith the Zea mays Ubiquitin 1 protein were identified from Brachypodiumdistachyon. Only one sequence that aligned with the Zea mays Ubiquitin 1protein was identified each from Setaria italic and Panicum virgatum. Anapproximately 2 kb DNA sequence upstream from a predicted translationalstart site (ATG) was determined to be the beginning of the putativepromoter sequence and used for expression characterization. Thepolynucleotide sequence alignments of the novel promoters that wereisolated from Panicum virgatum, Brachypodium distachyon and Setariaitalica were aligned to the ZM Ubi1 promoter and found to share lowlevels of sequence similarity across the 2 kb DNA region (FIGS. 2A-C).

The UBI coding sequence and putative promoter for the Panicum virgatum,Brachypodium distachyon and Setaria italica ubiquitin genes areindicated in FIGS. 35-38.

Example 3: Vector Construction

The four promoter sequences were commercially synthesized andincorporated into plasmid vectors as depicted in FIG. 3 (pDAB113091),FIG. 4 (pDAB113092), FIG. 5 (pDAB113066) and FIG. 22 (pDAB118238).Similarly four 3′UTR/transcription termination sequences werecommercially synthesized and incorporated into plasmid vectors asdepicted in FIG. 23 (pDAB118237), FIG. 24 (pDAB118207), FIG. 25(pDAB118208) and FIG. 26 (pDAB118209). The sequences were flanked by15-18 nucleotide homology fragments on both ends for seamless cloning(GeneArt® Seamless Cloning and Assembly Kit, Invitrogen, Carlsbad,Calif.) and type II restriction enzyme sites inserted for the isolationof promoter fragments. Seamless cloning compatible Zea mays Ubi1promoter (Christensen and Quail (1996) Transgenic Research. 5; 213-218;Christensen et al., (1992) Plant Molecular Biology. 18; 675-689) orOryzae sativa Actin promoter (McElroy et al., (1990) Plant Cell. 2;163-71), and PhiYFP (Shagin et al., (2004) Mol Biol Evol. 21; 841-50)coding sequence comprising the ST-LS1 intron (Vancanneyt et al., (1990)Mol Gen Genet. 220; 245-50), and St PinII or native 3′-UTR (An et al.,1989 Plant Cell. 1; 115-22.) fragments were obtained using PCR or typeIIrestriction enzymes. Finally, the promoter:: PhiYFP:: St PinII 3′-UTRfragments were assembled using seamless cloning to create transientexpression vectors (FIG. 6, pDAB113103; FIG. 7, pDAB113104; FIG. 8,pDAB113105; FIG. 9, pDAB113106; and, FIG. 10, pDAB113107; FIG. 27,pDAB120403; FIG. 28, pDAB118234, FIG. 29, pDAB118235; and FIG. 30,pDAB118236) for transient expression testing. These transient expressionvectors were integrated into a binary vector containing the Zm Ubi 1promoter and AAD-1 coding sequence (International Patent Publication No.2005107437) and Zm Lip 3′UTR (Paek et al., (1998) Molecules and Cells,8(3): 336-342). The resulting binaries were confirmed via restrictionenzyme digestion and sequencing reaction (FIG. 12, pDAB113117; FIG. 13,pDAB113118; FIG. 14, pDAB113119; FIG. 15, pDAB113120; FIG. 16,pDAB113121; FIG. 31, pDAB120400; FIG. 32, pDAB120404; FIG. 33,pDAB120401; and, FIG. 34, pDAB120402).

Example 4: Transient Expression Testing

Transient expression was tested using particle bombardment of immaturemaize (B104) embryos. Forty embryos were used per treatment in a Petriplate for bombardment. YFP image analysis was done after overnightincubation of particle bombardment. FIG. 19 shows YFP expression levelsobtained from the novel promoters. The data show that YFP expressionlevels obtained from the novel promoters (pDAB113103, pDAB113104, andpDAB113105) is comparable to the YFP expression levels obtained from theZM Ubi1 promoter (pDAB113106) and the OS Act 1 promoter (pDAB113107) asvisually observed under the microscope. Plant tissues were imaged on aLeica EL6000—mercury metal Halide™ microscope. Confocal and DifferentialInterference Contrast (DIC) images were captured using Chroma42003—ZsYellow 1™ filters.

Example 5: Transgene Copy Number Estimation Using Real Time TaqMan® PCR

The stable integration of the yfp transgene within the genome of thetransgenic Z. mays plants was confirmed via a hydrolysis probe assay.Stably-transformed transgenic Z. mays plantlets that developed from thecallus were obtained and analyzed to identify events that contained alow copy number (1-2 copies) of full-length T-strand inserts. Identifiedplantlets were advanced to the green house and grown.

The Roche Light Cycler480™ system was used to determine the transgenecopy number. The method utilized a biplex TaqMan® reaction that employedoligonucleotides specific to the yfp gene and to the endogenous Z. maysreference gene, invertase (Genbank Accession No: U16123.1), in a singleassay. Copy number and zygosity were determined by measuring theintensity of yfp-specific fluorescence, relative to theinvertase-specific fluorescence, as compared to known copy numberstandards.

A yfp gene-specific DNA fragment was amplified with one TaqMan®primer/probe set containing a probe labeled with FAM™ fluorescent dye,and invertase was amplified with a second TaqMan® primer/probe setcontaining a probe labeled with HEX™ fluorescence (TABLE 2). The PCRreaction mixture was prepared as set forth in TABLE 3, and thegene-specific DNA fragments were amplified according to the conditionsset forth in TABLE 4. Copy number and zygosity of the samples weredetermined by measuring the relative intensity of fluorescence specificfor the reporter gene, yfp, to fluorescence specific for the referencegene, invertase, as compared to known copy number standards.

TABLE 2 Forward and reverse nucleotide primer and fluorescent probes.Primer/Probe Sequence PhiYFP v3 Forward Primer (SEQ ID NO: 28)CGTGTTGGGAAAGAACTTGGA PhiYFP v3 Reverse Primer (SEQ ID NO: 29)CCGTGGTTGGCTTGGTCT PhiYFP v3 Probe (SEQ ID NO: 30)5′FAM/CACTCCCCACTGCCT/ MGB_BHQ_1/3′ Invertase Forward Primer (SEQ ID NO:31) TGGCGGACGACGACTTGT Invertase Reverse Primer (SEQ ID NO: 32)AAAGTTTGGAGGCTGCCGT Invertase Probe (SEQ ID NO: 33) 5′HEX/CGAGCAGACCGCCGTGTACTT/3BHQ_1/3′ (synthesized by Integrated DNATechnologies, Coralville, IA).

TABLE 3 Taqman ® PCR reaction mixture. Working Final Volume ComponentConcentration Concentration (μl) Water — — 0.5 Roche LightCyler 480 2X1X 5 Probes Master Mix PhiYFP v3 F 10 μM 400 nM 0.4 PhiYFP v3 R 10 μM400 nM 0.4 PhiYFP v3 Probe-FAM  5 μM 200 nM 0.4 Invertase F 10 μM 400 nM0.4 Invertase R 10 μM 400 nM 0.4 Invertase Probe - Hex  5 μM 200 nM 0.4Polyvinylpyrrolidone (PVP) 10% 0.1% 0.1 Genomic DNA template DilutedBioCel  ~10 ng/μL 2 DNA (~5 nglul) Total reaction volume — — 10.0

TABLE 4 Thermocycler conditions for PCR amplification. PCR Steps Temp (°C.) Time No. of cycles Step-1 95 10 minutes 1 Step-2 95 10 seconds 40 5835 seconds 72 1 second Step-3 40 seconds 1

Standards were created by diluting the vector, pDAB108706, into Z. maysB104 genomic DNA (gDNA) to obtain standards with a known relationship ofpDAB108706:gDNA. For example, samples having one; two; and four cop(ies)of vector DNA per one copy of the Z. mays B104 gDNA were prepared. Oneand two copy dilutions of the pDAB108706 mixed with the Z. mays B104gDNA standard were validated against a control Z. mays event that wasknown to be hemizygous, and a control Z. mays event that was known to behomozygous (Z. mays event 278; see PCT International Patent PublicationNo. WO 2011/022469 A2). A TaqMan® biplex assay that utilizesoligonucleotides specific to the AAD1 gene and oligonucleotides specificto the endogenous Z. mays reference gene, invertase, was performed byamplifying and detecting a gene-specific DNA fragment for AAD1 with oneTaqMan® primer/probe set containing a probe labeled with FAM fluorescentdye, and by amplifying and detecting a gene-specific DNA fragment forinvertase with a second TaqMan® primer/probe set containing a probelabeled with HEX™ fluorescence (TABLE 2). The AAD1 TaqMan® reactionmixture was prepared as set forth in TABLE 3, and the specific fragmentswere amplified according to the conditions set forth in TABLE 4.

The level of fluorescence that was generated for each reaction wasanalyzed using the Roche LightCycler® 480 Thermocycler according to themanufacturer's directions. The FAM™ fluorescent moiety was excited at anoptical density of 465/510 nm, and the HEX™ fluorescent moiety wasexcited at an optical density of 533/580 nm. The copy number wasdetermined by comparison of Target/Reference values for unknown samples(output by the LightCycler® 480) to Target/Reference values of fourknown copy number standards (Null, 1-Copy (hemi), 2-Copy (homo) and4-Copy). Results from the transgene copy number analysis of transgenicplants obtained via transformation with different promoter constructsare shown in TABLE 5. Only plants with 1-2 copies of the yfp transgenewere transferred to the greenhouse for further expression analyses.

TABLE 5 Transgene copy number estimation of the transgenic plantsobtained from promoter construct described herein and controlconstructs. Number of 1-2 Copies Construct Positive Events of yfppDAB113117 32 17 pDAB113118 26 13 pDAB113119 30 16 pDAB113120 43 10pDAB113121 36 19

Example 6: Expression of Genes Operably Linked to Ubiquitin PromotersProtein Extraction

T₀ plants were sampled at V4-5 using a leaf ELISA assays. Sample werecollected in 96-well collection tube plate, and 4 leaf disks (paper holepunch size) were taken for each sample. Two 4.5 mm BBs and 200 μLextraction buffer [1×PBS supplemented with 0.05% Tween®-20 and 0.05% BSA(Millipore Probumin®, EMD Millipore Corp., Billerica, Mass.)] were addedto each tube. For AAD1 extraction, the concentration of BSA wasincreased to 0.5%. Plates were processed in a KLECO bead mill at fullspeed for 3 minutes. Additional 200 μL of extraction buffer was added toeach tube followed by inversion to mix. Plates were spun for 5 minutesat 3000 rpm. Supernatant was transferred to corresponding wells in adeep well 96 stored on ice.

YFP and AAD1 ELISA Procedure

Nunc® 96-well Maxi-Sorp Plates (Thermo Fisher Scientific Inc., Rockford,Ill.) were used for ELISA. Plates were coated with mouse monoclonalanti-YFP capture antibody (OriGene Technologies Inc., Rockville, Md.).The antibody was diluted in PBS (1 μg/mL) and 150 μL of diluted PBS wasadded per well. The plates were incubated overnight at 4° C. Theovernight plates were kept at room temperature for 20-30 minutes beforewashing 4× with 350 μL of wash buffer [1×PBS supplemented with 0.05%Tween®-20 (Sigma-Aldrich, St. Louis, Mo.)]. Plates were blocked with 200μL per well of blocking buffer [1×PBS supplemented with 0.05% Tween®-20plus 0.5% BSA (Millipore Probumin®)] for a minimum of 1 hr at +37° C.followed by 4× washing with 350 μL of wash buffer (Tomtec QuadraWash™ 2,Tomtec, Inc., Hamden, Conn.).

For the YFP ELISA, Evrogen recombinant Phi-YFP 1 mg/mL (Axxora LLC,Farmingdale, N.Y.) was used as a standard. A 5-parameter fit standardcurve (between the 1 ng/ml and 0.125 ng/ml Standards) was used to ensureall data fall in the linear portion of the curve. 100 μL of standard orsample was added to the well. A minimum 1:4 dilution of sample in theAssay Buffer was used. Plates were incubated for 1 hr at RT on plateshaker (250 rpm; Titer Plate shaker) followed by 4× washing with 350 μLof wash buffer (Tomtec QuadraWash™ 2). About 100 μL of 1 μg/mL Evrogenrabbit polyclonal anti-PhiYFP primary antibody (Axxora) was added toeach well. Plates were incubated for 1 hr at room temperature on a plateshaker at 250 rpm followed by 4× washing with 350 μL of wash buffer(Tomtec QuadraWash™ 2). Next, 100 μL of anti-rabbit IgG HRP secondaryantibody (Thermo Scientific) diluted 1:5000 in Blocking/Assay buffer,which was added to each well. Plates were incubated for 1 hr at roomtemperature on plate shaker at 250 rpm followed by 4× washes with 350 μLof wash buffer (Tomtec QuadraWash™ 2). 100 μL of Pierce 1 Step Ultra TMBELISA (Thermo Scientific) substrate was added in the well with gentleshaking for 10 minutes. Reaction was stopped by adding 50 μL of 0.4NH₂SO₄. Absorbance was read at 450 nm with a 650 nm reference filter.

AAD1 expression levels were determined by ELISAs using kits from AcadiaBioSciences (Portland, Me.). The ELISAs were performed using multipledilutions of the extracts and using the reagents and instructionsprovided by the supplier. The protein levels were normalized using totalsoluble protein assay, performed using the 660 nm protein assay reagentsupplied by Thermo Scientific and following the supplier's instructions.

Example 7: Whole Plant YFP Image Analysis Exemplifying Stable Expressionof Genes Operably Linked to Ubiquitin Promoters

Whole plants that contained a low copy number of the binary plasmid weregrown in a greenhouse. Plant tissues were imaged on a LeicaEL6000—mercury metal Halide™ microscope. Confocal and DifferentialInterference Contrast (DIC) images were captured using Chroma42003—ZsYellow 1™ filters. Representative examples of stable expressionof YFP in callus and root tissue of transgenic T₀ maize plants obtainedfrom Z. mays embryos transformed with the Brachypodium distachyonUbiquitin1 C, Brachypodium distachyon Ubiquitin1, and Setaria italicaubiquitin 2 promoters described herein are presented in FIG. 20 to FIG.21, respectively. The promoters drove robust expression of the yfpcoding sequences both in callus (FIG. 20) and root (FIG. 21) planttissues.

Example 8: Whole Plant T₀ Stable Expression of Genes Operably Linked toUbiquitin Promoters

Additional data was produced from an ELISA analysis of the expressed YFPprotein. The ELISA analysis further confirmed that the novel promotersdrove robust expression of a transgene. The quantitative measurements ofYFP protein obtained from transgenic plants comprising novel promoterconstructs are shown in FIG. 17 and TABLE 6. The data show thatexpression of YFP protein in the plants containing the novel promoters(pDAB113117, pDAB113118, and pDAB113119) is several fold higher that YFPexpression obtained from the Os Act1 (Rice Actin1) promoter(pDAB113120). Comparatively, FIG. 18 and TABLE 7 show that similar levelof AAD1 expression was obtained from all the constructs. This isexpected because AAD1 is driven by the Zm Ubi1 promoter for all of theconstructs.

TABLE 6 Cross Species Ubiquitin Promoter T₀ Leaf YFP expression MeanStatistical Construct (ng/mg TSP) significance pDAB113121 144.00173 ApDAB113118 92.37256 AB pDAB113119 65.30393 B pDAB113117 55.24345 BpDAB113120 12.77181 B

Levels not connected by same letter are significantly different

TABLE 7 Cross Species Ubiquitin Promoter T₀ Leaf AAD1 expression MeanStatistical Construct (ng/mg TSP) significance pDAB113121 119.06932 ApDAB113118 109.19796 A pDAB113119 96.29021 A pDAB113117 85.40412 ApDAB113120 83.81594 A

Levels not connected by same letter are significantly different

Example 9: Whole Plant T₁ Stable Expression of Genes Operable Linked toUbiquitin Promoters and 3′UTRs

T₀ single transgene copy plants were backcrossed to wild type B104 cornplants to obtain T₁ seed. Hemizygous T₁ plants were used for analysis.Five events per construct and 5-10 plants per event for V4 and V12 leafexpression. Three events per construct and 3 plants per event were usedfor the other tissue type expression. Zygosity analysis was done forAAD1/YFP.

The quantitative measurements of YFP protein obtained from leaf tissueof T₁ transgenic plants comprising novel promoter constructs are shownin TABLE 8. The data confirmed the T₀ leaf expression results andfurther showed that consistent high expression of YFP protein wasobtained in the V4, V12 and R3 leaf tissue of the plants containing thenovel promoters (pDAB113117, pDAB113118, and pDAB113119). TABLE 8 alsoshows that there was several fold increase in the expression of YFPprotein when this novel promoters were used in combination with theirnative 3′UTRs (pDAB120400, pDAB120401, and pDAB120402) instead of PinII3′UTR (pDAB113117, pDAB113118, and pDAB113119). YFP protein expressionwas detected from the plants containing construct pDAB120404 confirmingthat novel promoter and 3′UTR used in this construct drive expression ofa transgene.

TABLE 8 Cross Species Ubiquitin Promoter and 3′UTR T₁ Leaf ExpressionMean YFP (ng/mg TSP) V4 V12 R3 Construct Event Leaf Leaf Leaf pDAB113117pDAB113117[1]-006 44.0 169.4 2108.3 pDAB113117 pDAB113117[1]-007 44.8181.4 2582.7 pDAB113117 pDAB113117[1]-008 79.6 322.8 4096.3 pDAB113117pDAB113117[1]-019 74.3 369.1 3420.3 pDAB113117 pDAB113117[1]-028 34.2168.2 2164.1 pDAB113118 pDAB113118[1]-005 33.6 148.8 2094.7 pDAB113118pDAB113118[1]-007 54.9 180.1 2171.4 pDAB113118 pDAB113118[1]-010 138.02748.2 pDAB113118 pDAB113118[1]-023 46.2 156.6 2216.8 pDAB113118pDAB113118[1]-025 41.7 132.9 2071.4 pDAB113119 pDAB113119[1]-001 133.1436.0 6744.0 pDAB113119 pDAB113119[1]-005 49.2 138.6 1772.9 pDAB113119pDAB113119[1]-011 54.6 133.9 1415.5 pDAB113119 pDAB113119[1]-013 129.11807.7 pDAB113119 pDAB113119[1]-028 38.5 129.9 1632.8 pDAB113120pDAB113120[1]-005 9.8 69.6 493.5 pDAB113120 pDAB113120[1]-010 24.3 74.5638.3 pDAB113120 pDAB113120[1]-014 17.2 79.7 552.4 pDAB113120pDAB113120[1]-023 13.2 55.4 372.2 pDAB113120 pDAB113120[1]-032 12.5 69.6233.7 pDAB113121 pDAB113121[1]-008 327.9 pDAB113121 pDAB113121[1]-011166.2 271.2 4472.6 pDAB113121 pDAB113121[1]-018 128.2 362.0 7116.3pDAB113121 pDAB113121[1]-023 112.2 309.1 6813.7 pDAB113121pDAB113121[1]-026 118.7 311.7 6300.7 pDAB120400 pDAB120400[1]-001640.8182 pDAB120400 pDAB120400[1]-002 339.24463 pDAB120400pDAB120400[1]-004 943.96511 pDAB120400 pDAB120400[1]-007 1653.7402pDAB120400 pDAB120400[1]-024 466.01906 pDAB120401 pDAB120401[1]-001833.04373 pDAB120401 pDAB120401[1]-011 471.9103 pDAB120401pDAB120401[1]-019 795.08285 pDAB120401 pDAB120401[1]-022 721.58288pDAB120401 pDAB120401[1]-025 696.94286 pDAB120402 pDAB120402[1]-010750.82185 pDAB120402 pDAB120402[1]-011 619.38603 pDAB120402pDAB120402[1]-014 618.98144 pDAB120402 pDAB120402[1]-030 625.84385pDAB120404 pDAB120404[1]-003 44.088479 pDAB120404 pDAB120404[1]-01347.464389 pDAB120404 pDAB120404[1]-014 52.204801 pDAB120404pDAB120404[1]-016 45.397854 pDAB120404 pDAB120404[1]-020 46.913279

High YFP protein expression was found in different tissue typesincluding cob, husk, kernel, pollen, root, silk and stem sampled fromthe transgenic corn plants containing novel Ubiquitin Promoters drivingYFP (Table 9). These data demonstrate that the novel promoters and3′UTRs claimed here drive high constitutive expression of transgene inplants and would be useful for biotechnological applications.

TABLE 9 Cross Species Ubiquitin Promoter T1 Expression in DifferentTissue Type Mean YFP (ng/mg TSP) Construct Event Cob Husk Kernel PollenV12 Root Silk Stem pDAB113117 pDAB113117[1]-006 3452.1 1164.3 1341.2397.1 2292.7 1405.0 7279.8 pDAB113117 pDAB113117[1]-007 2519.6 954.71410.9 414.3 2245.6 1974.3 6179.0 pDAB113117 pDAB113117[1]-019 8362.32280.8 2829.6 749.5 7112.4 4790.2 13044.1 pDAB113118 pDAB113118[1]-0052801.6 620.9 886.3 782.2 1136.1 636.7 1953.6 pDAB113118pDAB113118[1]-007 2339.2 524.9 725.1 376.1 1495.6 1271.0 2806.9pDAB113118 pDAB113118[1]-023 1302.1 491.8 716.8 435.3 1193.0 829.11522.9 pDAB113119 pDAB113119[1]-011 399.7 1025.9 942.2 2475.6 pDAB113119pDAB113119[1]-013 2238.1 572.0 1050.5 438.1 1311.2 539.7 2235.2pDAB113119 pDAB113119[1]-028 2013.9 536.4 1061.4 450.0 1166.3 826.91912.5 pDAB113120 pDAB113120[1]-005 1166.8 310.5 514.0 1704.1 169.7322.1 739.3 pDAB113120 pDAB113120[1]-023 1096.4 531.9 845.8 1433.9 268.4572.2 877.9 pDAB113120 pDAB113120[1]-032 1344.1 587.8 985.1 1252.3 187.6472.4 694.0 pDAB113121 pDAB113121[1]-011 6779.1 2942.3 3452.6 2022.65834.0 2881.7 7445.5 pDAB113121 pDAB113121[1]-023 4830.0 2689.8 1913.71641.8 2547.7 2453.8 8295.9 pDAB113121 pDAB113121[1]-026 8186.2 3889.34432.3 1432.0 2521.9 2182.5 7760.7

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to theextent they are not inconsistent with the explicit details of thisdisclosure, and are so incorporated to the same extent as if eachreference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention. The followingexamples are provided to illustrate certain particular features and/orembodiments. The examples should not be construed to limit thedisclosure to the particular features or embodiments exemplified.

What is claimed is:
 1. A nucleic acid vector comprising a promoter,wherein i) said promoter is operably linked to a polylinker sequence;ii) said promoter is operably linked to a non-ubiquitin-coding sequence;or iii) said promoter is operably linked to a sequence comprising apolylinker sequence and a non-ubiquitin-coding sequence, wherein saidpromoter comprises SEQ ID NO: 1 or a sequence that has at least 99%sequence identity with SEQ ID NO: 1 and can initiate transcription of anoperably linked coding sequence.
 2. The nucleic acid vector of claim 1wherein said promoter is less than 3 kb in length.
 3. The nucleic acidvector of claim 1 wherein said promoter consists of SEQ ID NO:
 1. 4. Thenucleic acid vector of claim 1 further comprising a sequence encoding aselectable marker.
 5. The nucleic acid vector of claim 4 wherein saidpromoter is operably linked to a transgene.
 6. The nucleic acid vectorof claim 5 wherein the transgene encodes a selectable marker or a geneproduct conferring insecticidal resistance, herbicide tolerance,nitrogen use efficiency, water use efficiency, or nutritional quality.7. The nucleic acid vector of claim 1 further comprising a 3′untranslated sequence comprising SEQ ID NO: 4 or a sequence that has atleast 99% sequence identity with SEQ ID NO: 4, wherein the 3′untranslated sequence is operably linked to said polylinker or saidcoding sequence.
 8. The nucleic acid vector of claim 1 furthercomprising a 5′ untranslated sequence comprising SEQ ID NO: 11 or asequence that has at least 99% sequence identity with SEQ ID NO: 11,wherein the 5′ untranslated sequence is inserted between, and operablylinked to, said promoter sequence and said polylinker or said codingsequence.
 9. The nucleic acid vector of claim 8 further comprising anintron sequence inserted after the 5′ untranslated sequence.
 10. Thenucleic acid vector of claim 9 wherein the intron sequence comprises SEQID NO:
 7. 11. The nucleic acid vector of claim 1 wherein the promoterconsists of SEQ ID NO: 40 or a sequence having at least 99% sequenceidentity with SEQ ID NO: 40, and said promoter being operably linked tosaid polylinker or said coding sequence, wherein said promoter caninitiate transcription of the operably linked coding sequence.
 12. Thenucleic acid vector of claim 1 wherein the promoter consists of SEQ IDNO: 15 or a sequence having at least 99% sequence identity with SEQ IDNO: 15, said promoter being operably linked to said polylinker or saidcoding sequence, wherein said promoter can initiate transcription of theoperably linked coding sequence.
 13. The nucleic acid vector of claim 12further comprising a 3′ untranslated sequence comprising SEQ ID NO: 4 ora sequence that has at least 99% sequence identity with SEQ ID NO: 4,wherein the 3′ untranslated sequence is operably linked to saidpolylinker or said coding sequence.
 14. A non-Brachypodium plantcomprising a Brachypodium promoter, wherein said Brachypodium promotercomprises SEQ ID NO: 1, or a sequence that has at least 99% sequenceidentity with SEQ ID NO: 1 operably linked to a coding sequence whereinsaid Brachypodium promoter can initiate transcription of the operablylinked coding sequence.
 15. The plant of claim 14 wherein said plant isselected from the group consisting of maize, wheat, rice, sorghum, oats,rye, bananas, sugar cane, soybean, cotton, sunflower, and canola. 16.The plant of claim 14 wherein said plant is Zea mays.
 17. The plant ofclaim 16 wherein the operably linked coding sequence is a transgene,further wherein said transgene is inserted into the genome of saidplant.
 18. The plant of claim 14 further comprising a 5′ untranslatedsequence comprising SEQ ID NO: 11 or a sequence that has at least 99%sequence identity with SEQ ID NO: 11, wherein the 5′ untranslatedsequence is inserted between, and operably linked to, said Brachypodiumpromoter and said coding sequence.
 19. The plant of claim 18 furthercomprising an intron sequence inserted after the 5′ untranslatedsequence.
 20. The plant of claim 19 wherein the intron sequencecomprises SEQ ID NO:
 7. 21. The plant of claim 18 further comprising a3′ untranslated sequence comprising SEQ ID NO: 4 or a sequence that hasat least 99% sequence identity with SEQ ID NO: 4, wherein the 3′untranslated sequence is operably linked to said coding sequence. 22.The plant of claim 14 wherein the Brachypodium promoter consists of SEQID NO: 40 or a sequence having at least 99% sequence identity with SEQID NO: 40 and can initiate transcription of the operably linked codingsequence further wherein said Brachypodium promoter is operably linkedto said coding sequence.
 23. The plant of claim 14 wherein theBrachypodium promoter consists of SEQ ID NO: 15 or a sequence having atleast 99% sequence identity with SEQ ID NO: 15 and can initiatetranscription of an operably linked coding sequence further wherein saidBrachypodium promoter is operably linked to said coding sequence. 24.The plant of claim 23 further comprising a 3′ untranslated sequencecomprising SEQ ID NO: 4 or a sequence that has at least 99% sequenceidentity with SEQ ID NO: 4, wherein the 3′ untranslated sequence isoperably linked to said coding sequence.
 25. A nucleic acid vectorcomprising a transcription terminator, wherein i) said terminator isoperably linked to a polylinker sequence; ii) said terminator isoperably linked to a non-ubiquitin-coding sequence; or iii) saidterminator is operably linked to a sequence comprising a polylinkersequence and a non-ubiquitin-coding sequence, wherein said transcriptionterminator comprises SEQ ID NO: 4 or a sequence that has at least 99%sequence identity with SEQ ID NO:
 4. 26. The nucleic acid vector ofclaim 25 wherein said transcription terminator is less than 1 kb inlength.
 27. The nucleic acid vector of claim 26 wherein saidtranscription terminator consists of SEQ ID NO: 4.